Modified nucleotide reagents

ABSTRACT

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/600,665, filed on May 19, 2017, which is a continuation-in-part ofU.S. patent application Ser. No. 15/357,958, filed on Nov. 21, 2016,which claims the benefit of U.S. Provisional Application No. 62/258,414,filed on Nov. 20, 2015, the disclosures of which are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

This application includes a Sequence Listing, as set forth in anASCII-compliant text file named“1407-00-012U03_2019-06-13_Seq_list_ST25.txt”, created on Jun. 13, 2019,and containing 136,130 bytes, which is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION

The development of novel modified nucleotide reagents, in particular thegeneration of nucleotide reagents containing fluorescent labels, hasincreased the power of nucleotide sequencing reactions, for examplenucleotide sequencing reactions that provide for the identification ofall four bases in a single reaction solution. Such methods have beenemployed in the “real-time” detection of incorporation events, where theact of incorporation gives rise to a signaling event that can bedetected. In particularly elegant methods, labeling components arecoupled to portions of the nucleotides that are removed during theincorporation event, eliminating any need to remove such labelingcomponents before the next nucleotide is added. See, e.g., Eid, J. etal. (2009) Science 323:133-138.

At the same time, however, the demands of next-generation sequencing,including whole-genome sequencing and resequencing, transcriptomeprofiling, epigenomic characterization, analysis of DNA-proteininteractions, and the like, require increased throughput at lower costper base sequenced. Higher throughput can impact the quality of thesequencing data obtained, however. For example, in any enzyme-mediated,template-dependent sequencing process, the overall fidelity,processivity, and/or accuracy of the incorporation process can havedirect impacts on sequence identification. In turn, lower accuracy mayrequire multiple fold coverage to identify a particular sequence with ahigh level of confidence. There is therefore a continuing need toincrease the performance of nucleotide sequencing reactions inanalytical systems. In particular, there is a continuing need to developmodified nucleotide reagents that have improved kinetic properties insingle-molecule real time sequencing reactions and that display otherdesirable characteristics.

SUMMARY OF THE INVENTION

The present disclosure addresses these and other needs by providing inone aspect a nucleotide compound represented by structural formula (I):

wherein

L is a nucleotide linker element comprising at least one affinitymodulating element;

P is a polyphosphate element;

Nu is a nucleoside element;

X is a multivalent central core element;

B″ is a terminal coupling element;

n is an integer from 1 to 4; and

o is 0 or 1.

In some embodiments, the at least one affinity modulating elementdecreases the K_(m) of a nucleotide-dependent enzyme for the nucleotidecompound or increases the enzymatic rate of the nucleotide-dependentenzyme.

In some embodiments, the at least one affinity modulating element is anaromatic spacer element or a shield element. More specifically, thearomatic spacer element can be a substituted or unsubstitutedmonocyclic, bicyclic, tricyclic, or tetracyclic aromatic moiety,including an anionic aromatic moiety.

In other embodiments, the at least one affinity modulating element is ashield element, including embodiments wherein the shield elementcomprises a plurality of side chains, including side chains comprising anegatively-charged component.

In some embodiments, the terminal coupling element comprises a biotinmoiety, such as a bis-biotin moiety.

Also provided are nucleotide reagent compositions comprising an avidinprotein and a nucleotide compound of the disclosure.

The disclosure still further provides labeled nucleotide analogscomprising at least a first avidin protein, at least a first nucleotidecompound, wherein the nucleotide compound comprises at least oneaffinity modulating element, and at least a first dye-labeled compound.Still further provided are polymerase enzymes, reaction mixtures andmethods for sequencing a nucleic acid template, and systems forsequencing nucleic acids.

While primarily described in terms of nucleic acid polymerases, andparticularly DNA polymerases, it will be appreciated that the approachof providing improved nucleotide compounds, dye-labeled compounds, andlabeled nucleotide analogs comprising those compounds can be usefullyapplied to other enzyme systems where one may wish to directly observethe enzyme reaction, in real time. Such enzyme systems include, forexample, other synthesizing enzymes, e.g., RNA polymerases, reversetranscriptases, ribosomal polymerases, as well as other enzyme systems,such as kinases, phosphatases, proteases, nucleases, ligases, and thelike.

Related compounds, analogs, enzymes, reaction mixtures, methods, andsystems have been disclosed in PCT International Application Nos. PCT/US2016/063176, PCT/US 2016/063177, and PCT/US 2016/063178, filed on Nov.21, 2016, the disclosures of which are incorporated herein by referencein their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate an exemplary nucleic acidsequencing process that can be carried out using aspects of theinvention.

FIG. 2A illustrates a bis-biotin-labeled dye component and twobiotin-labeled nucleotide components associated with an avidin proteinshield. FIG. 2B illustrates a bis-biotin-labeled nucleotide componentand two biotin-labeled dye components associated with an avidin proteinshield. FIG. 2C illustrates a bis-biotin-labeled dye component and abis-biotin-labeled nucleotide component associated with an avidinprotein shield. FIG. 2D illustrates a dye component labeled with twobis-biotin moieties associating with two avidin protein shields, each ofwhich is associated with a nucleotide component comprising a bis-biotin.

FIGS. 3A-3Z and 3A′-3O′ illustrate exemplary labeled nucleotide analogsof the disclosure.

FIGS. 4A-4C illustrate exemplary dye-labeled compounds lacking shieldelements.

FIGS. 5A-5M illustrate exemplary dye-labeled compounds of the disclosurecomprising shield elements.

FIG. 6A graphically illustrates an exemplary intermediate structure forincorporation into a labeled nucleotide analog of the disclosure. FIGS.6B-6D illustrate exemplary chemical structures corresponding to theintermediate of FIG. 6A.

FIG. 6E illustrates the chemical synthesis of a bis-biotin-labeled,four-donor dye (“D4”) shielded intermediate compound and a graphicalrepresentation of the molecule. FIG. 6F illustrates another graphicillustration (left) and chemical structure (right) of an exemplaryshielded four-donor dye intermediate compound used in the assembly ofthe instant labeled nucleotide analogs.

FIGS. 7A-7D outline exemplary pathways for the synthetic assembly oflabeled nucleotide analogs of the disclosure. FIG. 7E illustrates therelationship between the graphic representations of some of thedifferent intermediate components illustrated in FIGS. 7A-7D and thechemical structures of those components. FIG. 7F illustrates additionallabeled nucleotide analog structures and their assembly from nucleotideand dye-labeled intermediate components and avidin proteins. FIG. 7Gshows the chemical structure of an alternatively shielded intermediatecomponent comprising four shielded donor dyes and two azide groups(left). Also shown is a graphic representation of an exemplary labelednucleotide analog that can be generated from the intermediate component(right).

FIG. 8 depicts interactions with the1H-2,3-dihydroisoquinoline-8-sulfo-6-carboxylic acid (“DISC”) group inthe crystal structure of a mutant Φ29 polymerase with a DISC-containinghexaphosphate analog. The polymerase includes E375Y and K512Ysubstitutions.

FIG. 9 depicts interactions with DISC and SG1 groups in the crystalstructure of a mutant Φ29 polymerase with a hexaphosphate analog. Thepolymerase includes E375W, K512F, and L142R substitutions.

FIG. 10 depicts interactions with the DISC group in the crystalstructure of a mutant Φ29 polymerase with a DISC-containinghexaphosphate analog. The polymerase includes E375W, K512H, and K135Rsubstitutions.

FIG. 11A depicts interactions with the DSDC group in a model of a mutantΦ29 polymerase with a DSDC-containing hexaphosphate analog. Thepolymerase includes E375Y, D510R, and K512Y substitutions.

FIG. 11B depicts interactions with the DSDC group in a model of a mutantΦ29 polymerase with a DSDC-containing hexaphosphate analog. Thepolymerase includes K135R, E375Y, D510R, and K512Y substitutions.

FIGS. 12A and 12B illustrate a comparison of the accuracy of sequencingwith mononucleotide and dinucleotide analogs.

FIGS. 13A and 13B illustrate a comparison of the kinetics of sequencingwith mononucleotide and dinucleotide analogs.

FIG. 14A compares sequencing reactions performed with a dinucleotideanalog and with modified mononucleotide analogs, each labeled with dG.FIG. 14B displays the normalized interpulse distance values for thereactions of FIG. 14A.

FIGS. 15A-15C illustrate normalized interpulse distances, global rates,and merging errors for a dinucleotide analog and for various modifiedmononucleotide analogs.

FIG. 16A shows normalized interpulse distances for nucleotide analogswith various anionic aromatic spacers. FIG. 16B shows IPD distributioncurves and FIG. 16C shows normalized pulsewidths for the same analogs.

FIG. 17A shows IPD distribution curves for nucleotide analogs withincreasing numbers of side chains. FIG. 17B shows normalized IPD valuesfor the same analogs.

FIG. 18 shows IPD distribution curves and normalized IPD values (inset)for mononucleotide and dinucleotide analogs with an anionic aromaticspacer.

FIG. 19A graphically illustrates some exemplary analog structures of thedisclosure. FIG. 19B illustrates normalized interpulse distances, andFIG. 19C illustrates polymerization rates for various nucleotide analogsof the disclosure.

FIG. 20 illustrates side chain embodiments of the disclosure.

FIG. 21 illustrates shield element embodiments of the disclosure.

FIG. 22 illustrates an exemplary intermediate compound of thedisclosure.

FIGS. 23A-23J illustrate exemplary dye-labeled compounds of thedisclosure.

FIG. 24 illustrates an exemplary synthetic route for preparing adendrimer side chain substituent.

FIG. 25 illustrates an exemplary synthetic pathway for generation of adendrimer having bifunctional reactivity as a linker.

FIGS. 26-37 illustrate exemplary nucleotide reagent compounds of thedisclosure.

DETAILED DESCRIPTION OF THE INVENTION

Labeled nucleotide analogs are used in a wide variety of differentapplications. Such applications include, for example, the observation ofsingle molecules, such as single biomolecules, in real time as theycarry out reactions. For ease of discussion, such labeled nucleotideanalogs, and particularly the exemplary nucleotide analogs of theinstant disclosure, are discussed herein in terms of a preferredapplication: the analysis of nucleic acid sequence information, andparticularly, single molecule nucleic acid sequence analysis.

In the preferred application, single molecule primer extension reactionsare monitored in real-time, to identify the ongoing incorporation ofnucleotides into the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in apolymerase-mediated template dependent primer extension reaction ismonitored as it occurs. In preferred aspects, the template/polymeraseprimer complex is provided, typically immobilized, within an opticallyconfined region, such as a zero mode waveguide (ZMW), or proximal to thesurface of a transparent substrate, optical waveguide, or the like (seee.g., U.S. Pat. Nos. 6,917,726, and 7,170,050 and U.S. PatentApplication Publication No. 2007/0134128, the disclosures of which arehereby incorporated by reference herein in their entirety for allpurposes). The optically confined region is illuminated with anappropriate excitation radiation for the fluorescently labelednucleotides that are to be used. Because the complex is within anoptically confined region, or very small illumination volume, only thereaction volume immediately surrounding the complex is subjected to theexcitation radiation. Accordingly, those fluorescently labelednucleotides that are interacting with the complex, e.g., during anincorporation event, are present within the illumination volume for asufficient time to identify them as having been incorporated.

A schematic illustration of this sequencing process is shown in FIGS.1A-1B. As shown in FIG. 1A, an immobilized complex 102 of a polymeraseenzyme, a template nucleic acid, and a primer sequence are providedwithin an observation volume (as shown by dashed line 104) of an opticalconfinement, of e.g., a zero mode waveguide 106. As an appropriatenucleotide analog, e.g., nucleotide 108, is incorporated into thenascent nucleic acid strand, it is illuminated for an extended period oftime, corresponding to the retention time of the labeled nucleotideanalog within the observation volume during incorporation, whichproduces a signal associated with that retention, e.g., signal pulse 112as shown by the A trace in FIG. 1B. Once incorporated, the label thatwas attached to the polyphosphate component of the labeled nucleotideanalog, is released. When the next appropriate nucleotide analog, e.g.,nucleotide 110, is contacted with the complex, it too is incorporated,giving rise to a corresponding signal 114 in the T trace of FIG. 1B. Bymonitoring the incorporation of bases into the nascent strand, asdictated by the underlying complementarity of the template sequence,long stretches of sequence information of the template can be obtained.

As described in PCT International Publication No. WO 2009/145828A2,which is incorporated by reference herein in its entirety for allpurposes, the incorporation of specific nucleotides can be determined byobserving bright phases and dark phases which correspond, for example,to reaction steps in which a fluorescent label is associated with thepolymerase enzyme, and steps in which the fluorescent label is notassociated with the enzyme. Under some conditions, the polymerasereaction system will exhibit two slow (kinetically observable) reactionsteps, wherein each of the steps is in a bright phase. Under otherconditions, the system will exhibit two kinetically observable reactionsteps, wherein each of the steps is in a dark phase. Under still otherconditions, the system will exhibit four kinetically observable (slow)reaction steps, two slow steps in a bright phase and two slow steps in adark phase. Factors influencing the observed kinetics include the typeof polymerase enzyme, the polymerase reaction conditions, including thetype and levels of cofactors, and the reaction substrates.

The labeled nucleotide analogs disclosed herein, including theirnucleotide and dye-labeled components, comprise structural features thatmodulate the kinetics of the polymerase reaction to improve theperformance of the system. The improved performance of the instantnucleotide analogs thus provides advantages for the use of these analogsin various analytical techniques. In particular, the instant disclosureprovides labeled nucleotide analogs that in some cases, among otheradvantageous properties, display shortened IPDs (inter-pulse distances)during SMRT™ DNA sequencing. The polymerase rates with these analogs isaccordingly increased. By modulating the IPD, which is related to theconcentration of the analogs added for the DNA sequencing reaction, theconcentration of the analog can be reduced. Reduction of analogconcentration correspondingly reduces background noise derived fromdiffusion of the analog in the ZMW and thus improves the signal to noiseratio. Improvement in these, and other, parameters is particularlyimportant where sequencing instruments would otherwise require higherpowers of laser illumination. Reduction of laser power in turn reducesphoto-bleaching of the fluorophores and other related photo damage.

While the usefulness of the labeled nucleotide analogs of the inventionis illustrated with the description above of SMRT™ sequencing, it is tobe understood that these analogs, and their nucleotide compoundcomponents and dye-labeled compound components, can be used with anyappropriate enzymatic or binding reaction and will thus have broaderapplication in other analytical techniques. For example, the labelednucleotide analogs of the instant disclosure are also useful in themeasurement of any type of binding interaction, not just bindinginteractions that result in the reaction of the reagent. While inpreferred embodiments, such as single-molecule, real-time nucleic acidsequencing reactions and other nucleotide-dependent enzymatic reactions,the analogs serve as an enzyme substrate and are chemically altered as aresult of the interaction, in other embodiments, such as, for example,the binding of a labeled nucleotide analog to an antibody, a receptor,or other affinity agent, the analog remains unaltered as a result of theinteraction. Measurement of an enzymatic reaction, a bindinginteraction, or any other type of reaction or interaction, can beperformed using well-known fluorescence techniques and biochemicalprocesses. Examples of such techniques and processes includefluorescence resonance energy transfer (FRET), fluorescencecross-correlation spectroscopy, fluorescence quenching, fluorescencepolarization, flow cytometry, and the like.

The instant disclosure provides chemical formulae and specific chemicalstructures for the inventive nucleotide and dye-labeled compounds. Wherechemical moieties are specified by their conventional chemical formulae,written from left to right, they optionally equally encompass the moietywhich would result from writing the structure from right to left, e.g.,—CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is also intended tooptionally represent —S(O)₂NH—, etc. Moreover, where compounds can berepresented as free acids or free bases or salts thereof, therepresentation of a particular form, e.g., carboxylic or sulfonic acid,also discloses the other form, e.g., the deprotonated salt form, e.g.,the carboxylate or sulfonate salt. Appropriate counterions for salts arewell-known in the art, and the choice of a particular counterion for asalt of the invention is well within the abilities of those of ordinaryskill in the art. Similarly, where the salt is disclosed, this structurealso discloses the compound in a free acid or free base form. Methods ofmaking salts and free acids and free bases are well-known in the art.

The labeled nucleotide analogs of the instant disclosure are generallymeant to be used as substrates for polymerase enzymes, particularly inthe context of nucleic acid sequencing. Therefore, generally, anynon-natural base, sugar, or phosphate of the nucleotide or nucleosidephosphate can be included as a nucleotide or nucleoside phosphate of theinvention if the nucleoside phosphate is capable of acting as asubstrate for any natural or modified polymerase enzyme.

“Activated derivatives of carboxyl moieties”, and equivalent species,refers to a moiety on a component of the instant compounds or theirprecursors or derivatives or on another reagent component in which anoxygen-containing, or other, leaving group is formally accessed througha carboxyl moiety, e.g., an active ester, acyl halide, acyl imidazolide,etc. Such activated moieties can be useful in coupling the variouscomponents of the instant nucleotide and dye-labeled compounds andanalogs as they are assembled.

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which can be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturated alkylradicals include, but are not limited to, groups such as methyl,methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl”, unless otherwise noted, includes “alkylene”,“alkynyl”, and optionally, those derivatives of alkyl defined in moredetail below, such as “heteroalkyl”.

The term “heteroalkyl”, by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, and S, and wherein the nitrogen andsulfur atoms can optionally be oxidized and the nitrogen heteroatom canoptionally be quaternized. The heteroatom(s) O, N, S, P, and Si can beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms can be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like).

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen”, by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl”, “heteroalkyl”, “aryl”, and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Exemplary substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, SO₃R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, and—NO₂ in a number ranging from zero to (2m″+1), where m″ is the totalnumber of carbon atoms in such radical. R′, R″, R″, and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound or reagent ofthe invention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R″, and R′″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. Accordingly, from the above discussion of substituents,one of ordinary skill in the art will understand that the terms“substituted alkyl” and “heteroalkyl” are meant to include groups thathave carbon atoms bound to groups other than hydrogen atoms, such ashaloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like).

The substituents set forth in the paragraph above are referred to hereinas “alkyl group substituents”.

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, SO₃R′, —S(O)₂NR′R″, —NRSO₂R′, —CN,and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″, andR″″ are preferably independently selected from hydrogen, (C₁-C₅)alkyland heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When acompound or reagent of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringcan optionally be replaced with a substituent of theformula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—,—O—, —CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring can optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed can optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring canoptionally be replaced with a substituent of the formula—(CRR′)_(s)-J-(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and J is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″, and R′″ are preferably independentlyselected from hydrogen or substituted or unsubstituted (C₁-C₆)-alkyl.

The substituents set forth in the two paragraphs above are referred toherein as “aryl group substituents”.

When referring to components of the compounds and analogs of thedisclosure, the term “residue derived from,” refers to a residue formedby the reaction of a first reactive functional group on a firstcomponent (e.g., a multivalent central core element, a dye element, ashield element, a linker element, a terminal coupling element, and thelike) and a second reactive functional group on a second component(e.g., a multivalent central core element, a dye element, a shieldelement, a linker element, a terminal coupling element, and the like) toform a covalent bond. In exemplary embodiments, an amine group on thefirst component is reacted with an activated carboxyl group on thesecond component to form a residue including one or more amide moieties.Other permutations of first and second reactive functional groups areencompassed by the invention. For example, the copper-catalyzed reactionof an azide-substituted first component with an alkyne-substitutedsecond component results in a triazole-containing residue through thewell-known “click” reaction, as would be understood by those of ordinaryskill in the art. See Kolb et al. (2001) Angew. Chem. Int. Ed. Engl.40:2004; Evans (2007) Aus. J. Chem. 60:384.

In some embodiments, a copper-free variant of the click reaction can beused to couple the first and second reactive groups. See, e.g., Baskinet al. (2007) Proc. Natl Acad. Sci. U.S.A. 104:16793-97. For example, anazide-substituted first component can be reacted with a cycloalkyne,ideally a cyclooctyne, attached to the second component, in the absenceof a copper catalyst. Such so-called copper-free click reagents areavailable commercially. Examples of such cycloalkynes include, withoutlimitation, dibenzocyclooctyne-amine, bicyclo[6.1.0]non-4-yn-9-yl, ormonofluorinated cyclooctynes. Other coupling chemistries can also beusefully employed in the synthesis of the compounds of the instantdisclosure, as would be understood by those of ordinary skill in theart.

Copper-catalyzed and copper-free click reactions result in the followingexemplary linkages, including triazole and cycloalkyl-containingresidues. Such residues should therefore be considered within the scopeof any linker or other substructure of the compounds disclosed herein,wherever they occur.

In addition, variation in the above linkages, for example where thelengths of the alkyl linker groups are altered, or where heteroatoms orother intervening chemical moieties are substituted for the structuresshown, are envisioned where such substitution does not interfere withthe function of the linker group, as would be understood by those ofordinary skill in the art.

It should also be understood that the attachment sites for the first andsecond reactive functional groups in the just-described reactions cangenerally be reversed if so desired, depending on the situation. Forexample, in the case of a “click” reaction, the first component can beazide-substituted and the second component can be alkyne-substituted, asdescribed above, or the first component can be alkyne-substituted andthe second component can be azide-substituted. Such variation in thereactions is well within the skill of those in the art.

As used herein, a listed range of integers includes each of the integerswithin the range. For example, an integer from 2 to 6 includes theintegers 2, 3, 4, 5, and 6.

Labeled Nucleotide Analogs

The instant disclosure provides novel labeled nucleotide analogs for usein the measurement and analysis of enzymatic reactions and othermolecular recognition events, such as, for example, single-moleculereal-time sequencing of nucleic acids. The analogs comprise at least oneprotein shield, preferably an avidin protein shield, that is associatedwith at least one nucleotide compound and at least one dye-labeledcompound. As is well known in the art, avidin proteins, includingavidin, streptavidin, tamavidin, traptavidin, xenavidin, bradavidin,AVR2, AVR4, and homologs thereof, typically comprise four subunits, eachsubunit comprising one biotin binding site. An avidin protein can,therefore, tightly associate with one or more biotin-labeled nucleotidecompounds and with one or more biotin-labeled dye-containing compounds,thus creating a dye-labeled, protein-shielded, nucleotide analog,examples of which are described in U.S. Patent Application PublicationNo. 2013/0316912 A1, issued as U.S. Pat. No. 9,062,091, which isincorporated by reference herein in its entirety for all purposes.

As shown in FIGS. 2A-2C, the previously-described protein-shieldednucleotide analogs can include one or two dye components and one or twonucleotide components, depending on whether the dye component andnucleotide component has two or one biotin labels, respectively. Asshown in FIG. 2D, these analogs can also contain more than one avidinprotein shield, if either the nucleotide or dye component is designed tobridge multiple avidin tetramers. In the illustrations of FIGS. 2A-2D, astraight line between the dye or nucleotide component and an avidinsubunit represents the association of a single biotin label on thecomponent with one avidin subunit, whereas a semicircle contacting twoavidin subunits represents the association of a bis-biotin label on thecomponent with both avidin subunits.

Other examples of protected fluorescent reagent compounds, includingnucleotide analog compounds and multimeric protected fluorescent reagentcompounds, are described in U.S. Patent Application Publication Nos.2015/0050659 A1 and 2016/0237279 A1, the disclosures of which areincorporated by reference herein in their entireties for all purposes.

FIGS. 3A-Z and 3A′-3O′ illustrate the higher-order structure ofexemplary dye-labeled nucleotide analogs of the disclosure. For example,in FIG. 3A, the spherical component (330) represents a tetrameric avidinprotein shield, containing four binding sites for biotin. Thesemicircles (320) on the associated nucleotide and dye-labeled compoundcomponents represent bis-biotin moieties. The large, symmetric oblongglobules (310) represent dye elements, whereas the smaller, asymmetricglobules associated with the two-lobed structure (350) represent theside chains of a shield element, in this context serving as an affinitymodulating element within the nucleotide linker. The key-shaped groups(340) correspond to nucleotides (i.e., a nucleoside element plus apolyphosphate element).

FIG. 3E illustrates three other elements of the instant nucleotide anddye-labeled compounds. Specifically, the circular structure (360)represents an aromatic spacer element, and the six-lobed structure (370)represents a shield element. Each of these components can serve as anaffinity modulating element within the nucleotide linker of thenucleotide compound. The two-lobed structure (380) represents aphoto-protective shield element within the dye-labeled compound of theanalog. All of these components will be described in detail below.Exemplary chemical structures corresponding to each of the abovecomponents, and others, are also illustrated in FIGS. 7E and 7G.

The superstructures shown in FIGS. 3A-3Z and 3A′-3O′ illustrate the widestructural diversity available through the assembly of variousdye-labeled components and nucleotide-labeled components with one ormore avidin proteins. For example, the analogs can contain one (e.g.,FIGS. 3A, 3B, 3C, 3D, 3I, 3M, 3O, 3P, and 3F′), two (e.g., FIGS. 3E, 3G,3H, 3K, 3L, 3N, 3Q, 3R-3Z, 3A′-3E′, and 3G′-3O′), or three (e.g., FIGS.3F and 3J) avidin proteins, and even larger superstructures can beassembled, if desired. The analogs can contain nucleotide compounds withone (e.g., FIGS. 3E, 3F, 3G, 3J, 3L, 3O-3W, 3Y, 3Z, and 3A′-3O′), two(e.g., FIGS. 3A, 3B, 3C, 3D, 3H, 3I, 3K, 3M, 3N, and 3X), or morenucleoside elements. Other features, such as the use of a shield elementand/or an aromatic spacer element, for example an anionic aromaticspacer element, within the linker element of a nucleotide compound tomodulate the affinity and/or kinetics of an associated binding proteinor enzyme, as well as the shielding of the dye-labeled compound, eitherby direct coupling of the shield element and the dye or by including ashield element and/or side chain in the dye linker, can be included asdesired in a variety of combinations. Although the exemplary analogs ofFIGS. 3A-Z and 3A′-3O′ all include nucleotide and dye-labeled compoundsthat are attached through bis-biotin moieties, it should be understoodthat analogs can also be usefully assembled from compounds having singlebiotin moieties, such as in the structures of FIGS. 2A-2C.

Accordingly, the instant labeled nucleotide analogs can comprise anydesired number of avidin tetramers, nucleotide compounds, anddye-labeled compounds. For example, the analogs can comprise 1, 2, 3, 4,6, 10, or even more of each of these components, in any combination. Inspecific embodiments, the labeled nucleotide analogs comprise from 1 to4 of each of the components. In even more specific embodiments, thelabeled nucleotide analogs comprise 1, 2, or 3 avidin proteins, 1 or 2dye-labeled compounds, and 1 or 2 nucleotide compounds.

It is particularly advantageous to vary the number and type of dyeelements within a labeled nucleotide analog in order to provide thedesired colors and intensities of absorption and emission. Furthermore,as will be described in more detail below, the inclusion of dyes withoverlapping spectra within the dye-labeled compound of an analog complexenables the use of more advanced fluorescence techniques, such as, forexample, fluorescence resonance energy transfer, where an input opticalsignal is transferred from a “donor” dye within the structure to aneighboring “acceptor” dye, which then emits an optical signal at alonger wavelength than would occur from the donor fluorophore alone.Changing the number of fluorescent dyes within a single labelednucleotide analog additionally allows the intensity of the outputoptical signal to be modulated in useful ways if desired.

For example, when the labeled nucleotide analogs are used in DNAsequencing reactions, it can be useful to vary the color or otheroptical property of analog as a function of the nucleotide componentassociated with the analog. In particular, the nucleotide components ofthe analogs represented in FIGS. 3A-3D may differ only in the nature ofthe base group, e.g., dA, dG, dC, and dT. In combination with thatvariation, the dye components of the analogs can also be varied, forexample as shown by the different dye structures, 310, 312, 314, and316. Each of the nucleotide analogs is thus uniquely identifiable by thecolor and/or intensity of its optical output.

The dye-labeled compounds used to assemble the labeled nucleotideanalogs disclosed herein advantageously further include shield elements.As mentioned above and in FIGS. 2A-2D, protein-shielded dye-labeledpolymerase substrates have been described in U.S. Patent ApplicationPublication No. 2013/0316912 A1. Some of the dye-labeled componentsutilized in those analogs contained multiple acceptor dyes and donordyes, but the dye-labeled compounds themselves do not contain shieldelements. Examples of unshielded dye-labeled compounds are shown inFIGS. 4A-4C, where the acceptor dyes are designated “A”, the donor dyesare designated “D”, the terminal coupling element, in these examples abis-biotin, is designated by a semi-circle, and the dye compound linkerelement is designated by the line linking the different components ofthe structure. The small dots within the dye compound linker elements ofthe compounds illustrated in FIGS. 4B and 4C represent a triazolestructure or other residue resulting from a copper-catalyzed clickreaction, a copper-free click reaction, or other suitable couplingreaction.

The compounds of FIGS. 4A-4C can be compared to those illustrated inFIGS. 5A-5M, which represent dye-labeled compounds comprising one ormore shield elements. As was also shown in the structures of FIGS. 3A-3Zand 3A′-3O′, the side chains of shield elements within the compounds aredesignated as asymmetric globule structures in FIGS. 5A-5M.

The compounds of FIGS. 5A-5M illustrate the wide diversity of structuralvariation possible within the scope of the instant dye-labeledcompounds. Specifically, the compounds can include, without limitation,a single bis-biotin moiety (e.g., FIGS. 5A, 5B, and 5C) or a doublebis-biotin moiety (e.g., FIGS. 5D-5M); they can include unshieldedacceptors and directly shielded donors (e.g., FIGS. 5B, 5H, 5K, and 5L);they can include directly shielded acceptors and unshielded donors(e.g., FIGS. 5C, 5F, 5G, 5J, and 5M); they can include both directlyshielded acceptors and directly shielded donors (e.g., FIGS. 5A and 51);or they can include compounds with shield elements and/or side chains intheir dye compound linker elements (e.g., FIGS. 5D, 5E, 5F, 5G, and 5K).It should be understood that some compounds can include both shieldelements associated with an acceptor and/or a donor and shield elementsand/or side chains included within the dye compound linker element. Itshould also be understood that while the drawings of FIGS. 5A-5M canindicate different sizes, shapes, and/or locations of the dyes, shields,and linkers (e.g., in FIG. 5G where the side chains of the acceptorshield element are shown as being larger than the side chains of theshield elements within the dye linker), the size, shape, and/or locationof any component shown in the drawings should not be considered limitingof the actual structures, except as described explicitly herein.

Further diversity of dye-labeled compounds is illustrated in thenucleotide analogs shown in FIGS. 3E-3Z and 3A′-3O′, where thedye-labeled components include one-donor, one-acceptor compounds(“D1A1”) (FIG. 3I), two-donor, one-acceptor compounds (“D2A1”) (FIG.3M), two-donor, two-acceptor (“D2A2”) (FIGS. 3O-3Q and 3D′), four-donor,one-acceptor compounds (“D4A1”) (FIGS. 3H, 3K, and 3L), four-donor,two-acceptor compounds (“D4A2”) (FIGS. 3E-3G, 3J, 3N, 3R, 3Z, 3A′, and3N′), four-donor, four-acceptor compounds (“D4A4”) (FIGS. 3T, 3W, and3X), six-donor, two-acceptor compounds (“D6A2”) (FIGS. 3S, 3Y, 3C′, 3F′,and 3G′), six-donor, four-acceptor compounds (“D6A4”) (FIG. 3E′),eight-donor, two-acceptor compounds (“D8A2”) (FIGS. 3U, 3V, 3B′, 3H′,3I′, 3J′ (where the difference between the nucleotide compound of FIG.3I′ and FIG. 3J′ is the structure of the acceptor dye), and 3O′),ten-donor, four-acceptor compounds (“D10A4”) (FIGS. 3K′ and 3L′), andtwelve-donor, two-acceptor compounds (“D12A2”) (FIG. 3M′). As isapparent from the dye-labeled compound structures of these figures, thelocation and number of donor dyes, acceptor dyes, and shield elementscan advantageously be varied to obtain desired properties, includingbrightness, excitation and emission wavelength, photostability, andreaction kinetics in automated DNA sequencing reactions involving DNApolymerase, as will be described in further detail below.

In order to provide a more specific description of each of thesecomponents, the structural and functional properties of the differentnovel nucleotide and dye-labeled compounds, the assembly of thosecompounds into novel labeled nucleotide analogs, and the interactions ofthose novel analogs with wild-type and mutated DNA polymerases will bedescribed in detail in the following sections.

Nucleotide Compounds

As just described, the instant disclosure provides novel nucleotidecompounds useful in the assembly of labeled nucleotide analogs havingutility in the measurement and analysis of enzymatic reactions and othermolecular recognition events, such as, for example, the single-moleculereal-time sequencing of nucleic acids.

Accordingly, in one aspect, the disclosure thus provides compounds ofstructural formula (I):

wherein

L is a nucleotide linker element comprising at least one affinitymodulating element;

P is a polyphosphate element;

Nu is a nucleoside element;

X is a multivalent central core element;

B″ is a terminal coupling element;

n is an integer from 1 to 4; and

o is 0 or 1.

In general, a “linker” of the instant disclosure should be consideredbroadly to include any chemical moiety that provides a suitable covalentconnection between two or more components within a given compound. Alinker can be hydrophilic (e.g., tetraethylene glycol, hexaethyleneglycol, polyethylene glycol) or it can be hydrophobic (e.g., hexane,decane, etc.). Exemplary linkers include substituted or unsubstitutedC6-C30 alkyl groups, polyols (e.g., glycerol), polyethers (e.g.,poly(ethyleneglycol)), polyamines, amino acids (e.g., polyaminoacids),peptides, saccharides (e.g., polysaccharides) and combinations thereof.Such linkers typically comprise linear or branched chains, wherein thechain can be substituted at any suitable position, as desired, andwherein any carbon atom can be replaced by any suitable heteroatom. Alinker can comprise one or more alkyl, heteroalkyl, cycloalkyl,cycloheteroalkyl, aryl, or heteroaryl groups, if so desired.

The nucleotide linker element, L, of structural formula (I) morespecifically attaches the polyphosphate element of this structure to themultivalent central core element, if present, or directly to theterminal coupling element. In specific embodiments, the nucleotidelinker element comprises a C6-C20 alkyl group, optionally comprising,e.g., an amide bond, an ether bond, a phenylene group, a triazole group,another coupling residue, or the like, in any combination. In addition,in the instant nucleotide compounds of structural formula (I), thenucleotide linker element comprises at least one affinity modulatingelement, which can be an aromatic spacer element, a shield element, orboth an aromatic spacer element and a shield element.

As will be described in more detail below, the affinity modulatingelement of the instant nucleotide compounds can serve to enhance theinteraction between a labeled nucleotide analog of the invention and abiomolecule, such as an enzyme or binding protein. The affinitymodulating element can enhance the interaction through electrostatic,hydrophobic, steric, or other means. In an exemplary embodiment in whicha labeled nucleotide analog, comprising a nucleotide compound with anaffinity modulating element within the nucleotide linker element, isutilized in a single molecule nucleic acid sequencing technique, theaffinity modulating element can, in particular, enhance the interactionbetween the nucleotide analog and the DNA polymerase, thereby loweringthe K_(m) or otherwise influencing the kinetics of the sequencingreaction to achieve optimized residence time of the analog on thepolymerase or other desired behavior. In particular, and withoutintending to be bound by theory, it is believed that the affinitymodulating element, preferably an aromatic spacer element, such as ananionic aromatic spacer element, and/or a shield element, interactsfavorably with specific amino acid residues near the active site of thepolymerase enzyme and that these interactions are responsible for theimproved kinetic properties.

Accordingly, in some embodiments of the compounds of structural formula(I), the nucleotide linker element comprises an affinity modulatingelement, and in some of these compounds, the affinity modulating elementis an aromatic spacer element or a shield element. In some embodiments,the aromatic spacer element is a substituted or unsubstitutedmonocyclic, bicyclic, tricyclic, or tetracyclic aromatic moiety.

In more specific embodiments, the aromatic spacer element is representedby structural formula (II):

wherein

the A-ring and the B-ring is each independently an optionallysubstituted 5-7 atom cyclic structure, wherein at least one of theA-ring or the B-ring is aromatic; and

the A-ring or the B-ring optionally comprises at least one anionicsubstituent.

Even more specifically, the optional at least one anionic substituent is—SO₃H.

In other specific embodiments, the aromatic spacer element isrepresented by structural formula (IIA) or (IIB):

wherein

one of the A₁, A₂, A₃, and A₄ groups is

and the other groups are —CH₂— or a bond; and

R₁ is H or an anionic substituent and R₂ is H or an anionic substituent.

More specifically, the aromatic spacer element can be represented bystructural formula (IIC) or (IIC′):

wherein

R₁ is H or an anionic substituent.

In some alternative embodiments, the aromatic spacer element can berepresented by structural formula (IV):

wherein

R₁ is H or an anionic substituent.

According to some more specific nucleotide compound embodiments, the atleast one affinity modulating element is an anionic aromatic spacerelement. Still more specifically, the anionic aromatic spacer element isa substituted bicyclic or tricyclic anionic aromatic moiety. Even morespecifically, the anionic aromatic spacer element is represented bystructural formula (II):

wherein

the A-ring and the B-ring is each independently a 5-7 atom cyclicstructure, wherein at least one of the A-ring or the B-ring is aromatic;and

the A-ring or the B-ring comprises at least one anionic substituent. Insome of these embodiments, the at least one anionic substituent is—SO₃H. In some of these embodiments, the anionic aromatic spacer elementis represented by structural formula (IIA) or (IIB):

wherein

one of the A₁, A₂, A₃, and A₄ groups is

and the other groups are —CH₂—; and

R₁ is the at least one anionic substituent and R₂ is H or the at leastone anionic substituent, including embodiments wherein the anionicsubstituent is —SO₃H. In some of these embodiments, the anionic aromaticspacer element is represented by structural formula (IIC):

In some embodiments, the aromatic spacer element optionally comprises atleast one anionic substituent or at least two anionic substituents. Morespecifically, the optional anionic substituent can be —SO₃H.

In some embodiments, the aromatic spacer element can be represented byone of the following structural formulae:

wherein

R₁, R₂, R₃, R₄, R₅, and R₆ are independently H or an anionicsubstituent.

In more specific embodiments, the aromatic spacer element can berepresented by one of the following structural formulae:

Even more specifically, the aromatic spacer element can be representedby one of the following structural formulae:

In some embodiments of compounds of structural formula (I), thenucleotide linker element comprises a shield element. As describedabove, a shield element can serve as an affinity modulating element inthe instant nucleotide compounds, thus modulating the interactionsbetween the nucleotide compound and an associated enzyme or bindingprotein. The exact structure of the shield element is not believed to becritical, so long as the structure is large enough to modulate contactsbetween the labeled analog and a protein, or other molecule of interestthat binds to the analog. As disclosed herein, shield elements can leadto improved kinetic and/or other properties in nucleotide analogscontaining these structures, in particular through their interactionswith an enzyme, such DNA polymerase, or a binding protein. In thenucleotide compounds of structural formula (I) disclosed herein, theshield element does not comprise a protein.

In some embodiments, the shield element of the instant nucleotidecompounds preferably comprises a shield core element that providesmultivalent attachment sites for shield element side chains, where theshield element side chains provide the primary bulkiness or chargedensity of the shield element moiety and are thus believed to beresponsible for the advantageous interactions with nucleotide-bindingproteins.

Accordingly, the shield elements can in some embodiments comprise asuitable core structure that provides for the attachment of a pluralityof side chains to the shield element core. In specific embodiments, theshield element comprises the structure:

wherein each y is independently an integer from 1 to 6.

In some embodiments, the shield core elements provide a “layered”structure, where each linker element includes more than one shieldelement core.

The side chains attached to the different shield element cores canoptionally be different types of side chain, if desired. The use ofdifferent side chains in the different layers can provide for differentmicroenvironments within the shield element. The different layers can,for example, comprise pairs of neutral or negatively charged groups,depending on the desired behavior and the intended use of the shieldedcompound.

Exemplary shield elements usefully incorporated into the nucleotidecompounds of the instant disclosure include the following non-limitingstructures:

It should be understood that these groups can be inserted within anucleotide linker element, or other component of the nucleotidecompound, in any orientation, as would be understood by those ofordinary skill in the art. The nucleotide linker element preferablyfurther comprises a short alkyl or cycloalkyl group, such as, forexample, a hexyl or cyclohexyl group, to link the shield element orelements to the rest of the structure, but other moieties can besuitably employed for this purpose. For example, the linker element canbe chosen from any of the linkers described herein. The linker elementcan, in more specific embodiments, comprise a triazole.

In this regard, it should be understood that the shield elements are, insome embodiments, synthetically assembled into a nucleotide linkerelement using “click” reactions, or “copper-free click” reactions, as isdescribed, for example in U.S. Patent Application Publication No.2015/0050659 A1. The intermediate components are therefore preferablylabeled with azide groups and acetylene groups that react with oneanother to form a triazole structure. It should also be understood,however, that other methods of attachment can be used to generate theinstant analogs within the scope of the instant invention, as would beunderstood by those of ordinary skill in the art.

Some shield element structures can include three, four, or even more“layers” of side chains, for example as shown in the following formulae:—Sh(R₁)₂—Sh(R₂)₂—Sh(R₃)₂—; and—Sh(R₁)₂—Sh(R₂)₂—Sh(R₃)₂—Sh(R₄)₂;

where “Sh” is a shield core element, such as, for example,

and “R₁”, “R₂”, “R₃”, and “R₄” are side chains. It should be understoodthat the “R₁”, “R₂”, “R₃”, and “R₄” side chain groups can be the same ordifferent side chains, in any combination, as desired to achieveimproved kinetic or other properties of the instant labeled nucleotideanalogs. The shield element is attached to the linker element throughthe Sh group from either end of the shield element structure in theseexamples.

As is true of the shield elements generally, the exact structures of theside chain components of the shield elements are not believed to becritical, so long as they are large enough to provide the desiredeffects. In some embodiments, the side chains comprise polyethyleneglycol (PEG). In specific embodiments, the polyethylene glycol sidechains comprise polyethylene glycol with from 3 to 20 repeating ethyleneoxide units. In more specific embodiments, the polyethylene glycol sidechains comprise polyethylene glycol with from 4 to 10 repeating ethyleneoxide units. In some embodiments, the side chains comprise anegatively-charged component, such as, for example, a componentcomprising a sulfonic acid. In some embodiments, the side chainscomprise a combination of polyethylene glycol and another component,such as, for example a negatively-charged component.

The side chains can additionally comprise a core structure that providesfor branching within the side chains. In some embodiments, the sidechain comprises a substituted phenyl group. In specific embodiments, theside chain comprises the structure:

wherein each x is independently an integer from 1 to 6. In more specificembodiments, each x is independently an integer from 1 to 4.

The side chain can, in some embodiments, comprise a dendrimer. Adendrimer (or “dendron”) is a repetitively branched molecule that istypically symmetric around the core and that can adopt a sphericalthree-dimensional morphology. See, e.g., Astruc et al. (2010) Chem. Rev.110:1857. Incorporation of such structures into the shield elements ofthe instant compounds provides for advantageous properties through themodulation of contacts between the labeled nucleotide analog and one ormore biomolecules associated with the nucleotide analog. Refinement ofthe chemical and physical properties of the dendrimer through variationin primary structure of the molecule, including potentialfunctionalization of the dendrimer surface, allows the functionalproperties of the nucleotide analog to be adjusted as desired.Dendrimers can be synthesized by a variety of techniques using a widerange of materials and branching reactions, including those describedbelow, as is well-known in the art.

Exemplary dendimer structures usefully incorporated into the side chainsof the instant molecules include the following:

The structural and functional properties of the dendrimer sidechainsused in the instant compounds can be tuned by, for example, variation in(a) chain lengths and types, (b) position and degree of branching, and(c) end group presentations (neutral or charged, hydrophobic orhydrophilic groups, etc.).

In some embodiments, at least one side chain comprises a peptide chain.

In some embodiments, at least one side chain comprises a polysaccharide.

Non-limiting side chain examples include the following structures:

(corresponding to PEG7) and polyethylene glycols with other numbers ofrepeating unit;

Some side chain embodiments can include combinations of any of the abovecomponents, such as, for example, the following combination of apolyethylene and a negatively-charged side chain:

In some embodiments, the molecular weight of the side chain is at least300, 350, 400, 450, or even higher. In preferred embodiments, themolecular weight of the side chain is at least 300.

In preferred embodiments of the compounds of structural formula (I), thenucleotide linker element comprises both an anionic aromatic spacerelement and a shield element, where these elements have the definitionsprovided herein.

The polyphosphate element of structural formula (I) comprises apyrophosphate or a higher homologue of phosphate, such as a 3-mer,4-mer, 5-mer, 6-mer, 7-mer, 8-mer, or the like. The polyphosphateelement thus generally comprises from 2 to 10 phosphates. In preferredembodiments, the polyphosphate element comprises 4, 5, 6, 7, or 8phosphates. In some embodiments, a methylene moiety, NH moiety, or Smoiety can bridge two or more of the phosphorus atoms, replacing the POPlink with a PCH₂P link, a PNHP link, a PSP link, or the like. Thepolyphosphate element can be further modified if desired, for example bysubstitution of any of the other oxygen atoms with carbon or anotherheteroatom or by alkylation or other similar modification of any of thenon-bridging oxygens.

The nucleotide compounds of the instant disclosure further comprise oneor more nucleoside elements. As previously described, the nucleosideelement is responsible for recognition of the analog by an enzyme, suchas DNA polymerase, during an enzymatic reaction, such as a sequencingreaction. As is known in the art, nucleosides contain nucleobases. Inaddition to the naturally occurring nucleobases of ribonucleic acids anddeoxyribonucleic acids, i.e., adenine, cytosine, guanine, thymine, anduracil, the nucleotide compounds and analogs of the invention canoptionally include modified bases. For example, the nucleoside elementsdescribed herein can comprise at least one modified base moiety which isselected from the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-d-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

Typically, the nucleoside elements described herein can comprise eitherribose or deoxyribose. In some embodiments, the nucleoside elements cancomprise a modified sugar moiety selected from the group including, butnot limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

The nucleoside elements of the instant nucleotide compounds and analogspreferably comprise adenosine, guanosine, thymidine, uridine, orcytidine, and are preferably deoxyribose nucleosides, e.g., dA, dG, dT,or dC.

The multivalent central core element of structural formula (I) is anoptional component of the structure that enables a plurality ofpolyphosphate elements and nucleoside elements to be attached to thenucleotide compound. As is clear from the structure of formula (I), themultivalent central core element, when present, also serves as anattachment site for the terminal coupling element.

In some embodiments, the multivalent central core element comprises apolyamine moiety. Polyamines can be readily reacted with appropriateelectrophilic reagents, such as electrophilic nucleotide linkerelements, and the like, to generate nucleotide compounds or theirintermediates. It should be understood that the order of such reactionscan be varied, depending on the desired outcome, as would be understoodby those of ordinary skill in the art. Non-limiting examples ofpolyamines usefully employed in the multivalent central core elements ofthe instant disclosure include the following:

The skilled artisan would understand, however, that other polyamines canbe readily utilized in the nucleotide compounds of the instantdisclosure.

In specific embodiments, the multivalent central core element comprisesa substituted cyclohexane, more specifically a1,3,5-triamino-cyclohexane.

In other specific embodiments, the multivalent central core elementcomprises a substituted 1,3,5-triazine.

In still other specific embodiments, the multivalent central coreelement comprises a substituted benzene.

In some embodiments the multivalent central core element comprises anether linkage. In some embodiments, the multivalent central core elementcomprises an acyl linkage. Examples of such ether and acyl-linkedcentral core elements include the following structures:

These structures can be incorporated into the instant nucleotidecompounds as described in detail below and in U.S. Patent ApplicationPublication No. 2015/0050659 A1. In particular, ether-linked centralcore elements can be modified with acetylene-containing groups,including cycloalkyne-containing groups, and the acetylene groups canthen be coupled to azide-containing reagents using “click” chemistry or“copper-free click” chemistry. Likewise, carboxylate-containing centralcore elements can be activated using suitable reagents, and theactivated acyl groups can then be coupled to appropriate nucleophilicreagents as desired. Alternatively, or in addition, the central coreelements can be activated using azide-containing groups, and thosegroups can be coupled to acetylene-containing reagents, includingcycloalkyne-containing reagents, using “click” chemistry or “copper-freeclick” chemistry. Such reactions are well understood by those ofordinary skill in the art.

The nucleotide compounds of structural formula (I) still furthercomprise a terminal coupling element. In some embodiments, the terminalcoupling element comprises biotin. As is well known in the art, biotinis bound with high affinity by avidin proteins such as avidin,streptavidin, and the like. In preferred embodiments, the terminalcoupling element comprises bis-biotin. The linker coupling the twobiotin moieties in a bis-biotin terminal coupling element can be anysuitable linker, including the linkers described above. The linkerpreferably includes a multivalent central core element, such as thestructures described above, both to couple the two biotin moieties toone another and to serve as an attachment point for the terminalcoupling element to the rest of the nucleotide compound.

Exemplary terminal coupling elements comprising bis-biotin include thefollowing structures:

In embodiments of the nucleotide compounds of structural formula (I), nis an integer from 1 to 4, and o is 0 or 1. As should be clear from thestructure, when n is 1, a multivalent central core element need not beincluded, so o is preferably 0. In addition, it should be understoodthat when n is 2 to 4, a multivalent central core element is preferablyincluded in the compound, so o should be 1. In specific embodiments, nis 2 and o is 1. In other specific embodiments, n is 1 and o is 0.

In preferred embodiments, the nucleotide compounds of the instantdisclosure, whether comprising an aromatic spacer element, a shieldelement, or both an aromatic spacer element and a shield element as theaffinity modulating element, do not contain a fluorescent dye or anyother directly detectable label.

As should be understood from the instant disclosure, the terminalcoupling element of the nucleotide compounds of structural formula (I)typically mediates association of the nucleotide compound with othercomponents of the instant labeled nucleotide analogs. For example, andas will be described in detail below, where the terminal couplingelement is a biotin or bis-biotin, the nucleotide compound can associatenon-covalently with an avidin protein with high affinity. In someaspects, the disclosure thus further provides compositions comprising anucleotide compound of structural formula (I) and an avidin protein. Inthese compositions, it should be understood that the terminal couplingelement is not covalently modified by the association of the nucleotidecompound with the avidin protein shield, and the composition thusdistinctly comprises the original nucleotide compound and the avidinprotein shield as separate molecular entities.

In another aspect of the disclosure, however, it should be contemplatedthat the terminal coupling element of a nucleotide compound may comprisea reactive functional group that can be covalently bound to acomplementary reactive group on a second component, for example on anappropriately modified linker element, shield element, or dye-labeledcompound. Unlike the just-described non-covalent compositions, suchreactions generate a new molecular entity connected by a residue derivedfrom the reactive group of each component. As described elsewhere in thespecification, such residues can comprise, for example, an amide moietyderived from an amine group and an appropriately activated carboxylgroup or a residue resulting from a click reaction.

In yet another aspect of the disclosure are provided methods ofsynthesis of the instant nucleotide compounds, including nucleotidecompounds of structural formula (I), and their intermediates. Suchmethods can comprise the step of reacting any of the intermediatecompounds illustrated throughout the specification with a secondintermediate compound to generate a nucleotide compound or intermediateof the invention. Exemplary synthetic pathways are illustrated in thereaction schemes below, in the Examples, and in the accompanyingdrawings.

Dye-Labeled Compounds

In yet another aspect, the disclosure provides dye-labeled compounds foruse in generating the instant labeled nucleotide analogs.

In embodiments according to this aspect of the disclosure, thedye-labeled compound comprises:

a donor dye;

an acceptor dye;

a shield element;

a terminal coupling element; and

a dye compound linker element;

wherein the dye compound linker element covalently connects the terminalcoupling element to the donor dye, the acceptor dye, or the shieldelement.

In specific embodiments, the acceptor dye or the donor dye is directlycoupled to the shield element.

In other embodiments, the dye-labeled compound is a compound ofstructural formula (IIIA), (IIIB), (IIIC), (IIID), or (IIIE):

wherein

each L′ is independently a dye compound linker element;

each S is independently a shield element;

each A is independently an acceptor dye;

each D is independently a donor dye;

each B″ is independently a terminal coupling element;

each p is independently 0 or 1; and

each r is independently an integer from 0 to 8;

wherein the compound comprises at least one shield element, at least oneacceptor dye, and at least one donor dye.

In specific embodiments, the at least one acceptor dye or the at leastone donor dye is directly coupled to the at least one shield element.

In other specific embodiments, each r is independently an integer from 0to 4.

In even more specific embodiments of the compounds of structural formula(IIIA), (IIIB), (IIIC), (IIID), and (IIIE), each r is independently 1 or2.

In any of the dye-labeled compound embodiments it should be understoodthat the compounds can comprise more than one donor dye, more than oneacceptor dye, and/or more than one shield element. In specificembodiments, the compound comprises at least two donor dyes, and in someof those embodiments each donor dye is directly coupled to a donorshield element. More specifically, the compound can comprise at leastfour donor dyes, and in some of those embodiments each donor dye isdirectly coupled to a donor shield element. Even more specifically, thecompound can comprise at least six donor dyes, at least eight donordyes, at least ten donor dyes, or even at least twelve donor dyes. Insome of these embodiments, each donor dye can be directly coupled to adonor shield element.

In some specific embodiments, the compound comprises at least twoacceptor dyes, and in some of those embodiments each acceptor dye isdirectly coupled to an acceptor shield element. More specifically, thecompound can comprise at least four acceptor dyes, and in some of thoseembodiments each acceptor dye can be directly coupled to an acceptorshield element.

In some embodiments, the compound comprises at least two donor dyes andat least two acceptor dyes. In more specific embodiments, each donor dyecan be directly coupled to a donor shield element and/or each acceptordye can be directly coupled to an acceptor shield element. In someembodiments, the compound comprises at least four donor dyes and atleast two acceptor dyes, at least six donor dyes and at least twoacceptor dyes, at least eight donor dyes and at least two acceptor dyes,at least ten donor dyes and at least two acceptor dyes, or even at leasttwelve donor dyes and at least two acceptor dyes.

In some embodiments, the compound further comprises a shield element ora side chain element attached to one or more dye compound linkerelements without also being attached to a donor or acceptor dye. Inparticular, the shield element or side chain element can be attachedwhere two dye compound linker elements are coupled, thus positioning theshield element or side chain element between different dye groupsattached to different dye compound linker elements.

In still other embodiments, the dye-labeled compound is a compound ofstructural formula (IIIF):

wherein

each L′ is independently a dye compound linker element;

each S is independently a shield element;

each A is independently an acceptor dye;

each D is independently a donor dye;

each B″ is independently a terminal coupling element;

each p is independently 0 or 1; and

each r′ is independently an integer from 0 to 4;

wherein the compound comprises at least one shield element, at least oneacceptor dye, and at least one donor dye.

In more specific embodiments of the compound of structural formula(IIIF), each r′ is independently an integer from 0 to 2.

In more specific embodiments of the compound of structural formula(IIIF), each r′ is independently 0 or 1.

In yet other embodiments, the dye-labeled compound is a compound ofstructural formula (IIIG):

wherein

each L′ is independently a dye compound linker element;

each S is independently a shield element;

each Dye is independently either an acceptor dye or a donor dye;

each B″ is independently a terminal coupling element;

each p is independently 0 or 1;

each r″ is independently an integer from 0 to 8;

s is an integer from 1 to 6; and

t is 0 or 1;

wherein the compound comprises at least one shield element, at least oneacceptor dye, and at least one donor dye.

In more specific embodiments of the compound of structural formula(IIIG), each r″ is independently an integer from 0 to 4 or from 0 to 2.

In other more specific embodiments of the compound of structural formula(IIIG), each r″ is independently 0 or 1.

In some embodiments of the compound of structural formula (IIIG), s isan integer from 1 to 4.

In some embodiments of the compound of structural formula (IIIG), thecompound comprises at least two donor dyes, at least four donor dyes, atleast six donor dyes, at least eight donor dyes, at least ten donordyes, or at least twelve donor dyes. In other more specific embodimentsof the compound of structural formula (IIIG), the compound comprises atleast two acceptor dyes or at least four acceptor dyes. In still othermore specific embodiments of the compound of structural formula (IIIG),the compound further comprises at least two shield elements, at leastfour shield elements, or even more shield elements. In some of theseembodiments, the shield elements are directly coupled to a donor dye oran acceptor dye.

By “directly coupled” it should be understood that the donor or acceptordye and the shield element are covalently attached to one another withno intervening functional components. The direct coupling can include,however, short linker groups, for example amide bonds, ether linkages,short alkyl chains, and the like, that do not significantly separate theshield element from the dye.

The donor dye and the acceptor dye of the instant dye-labeled compoundsare preferably chromophores that are capable of resonance energytransfer between one another. In this regard, a pair of dyes areconsidered donor and acceptor dyes when the donor dye in anelectronically excited state can transfer energy to the acceptor dyethrough a radiative or non-radiative energy transfer process. Forexample, processes in which a photon is emitted and those involvinglong-range electron transfer are included within the meaning ofresonance energy transfer. Resonance energy transfer typically ariseswhen the distance between the donor dye and the acceptor dye is small,when the emission spectrum of the donor dye and the excitation spectrumof the acceptor dye overlap sufficiently, and when the dipole moments ofthe donor emission and acceptor excitation are relatively aligned withone another. Examples of FRET-labeled nucleotides and donor-acceptorpairing are provided in U.S. Patent Application Publication Nos.2010/0255488 and 2012/0058469, the full disclosures of which are herebyincorporated by reference herein in their entirety for all purposes.

The donor dye and the acceptor dye of the instant dye-labeled compoundsare preferably fluorescent dyes. The dyes preferably have excitation andemission spectra in the visible region of the electromagnetic spectrum,although the dyes can in some embodiments have excitation and emissionspectra in the infrared range. Any of the dyes set forth herein can be acomponent of a FRET pair as either the donor or acceptor. Conjugating adonor dye and an acceptor dye through reactive functional groups on thedonor dye, the acceptor dye, and any necessary shield elements and/ordye compound linker elements, is well within the abilities of those ofskill in the art in view of the instant disclosure.

A wide variety of fluorophores are readily available and applicable tothe dye-labeled compounds of the invention and include fluorescein, orrhodamine based dyes, cyanine dyes and the like. A variety of such dyesare commercially available and include the Cy dyes available from GEHealthcare (Piscataway, N.J.), such as Cy3, Cy5, and the like, or theAlexa family of dyes available from Thermo Fisher Scientific Inc., suchas Alexa 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660,680, 700, and 750. These fluorophores can be present as individualfluorophores or they can be present in interactive pairs or groups,e.g., as fluorescent resonant energy transfer (FRET) pairs.

In preferred embodiments, the fluorescent dye is a cyanine dye, forexample any of the cyanine dyes disclosed in PCT InternationalPublication No. 2012/027618; U.S. Patent Application Publication No.2012/0058469; U.S. Patent Application Publication No. 2012/0058482; andU.S. Patent Application Publication No. 2012/0052506; the disclosures ofeach of which are incorporated herein by reference in their entiretiesfor all purposes. Additional long-wavelength heteroarylcyanine dyesusefully incorporated into the instant dye-labeled compounds aredisclosed in U.S. Patent Application Publication No. 2014/0005404 A1,the full disclosure of which is hereby incorporated by reference hereinfor all purposes.

The term “cyanine”, as used herein, thus refers to polymethine dyes suchas those based upon the cyanine, merocyanine, styryl and oxonol ring.Cyanine dyes include, for example, CY3, CY3.5, CY5 and CY5.5 type dyes.

Exemplary cyanine dyes have the formula:

wherein the A-ring and B-ring are independently selected frommonocyclic, bicyclic or polycyclic aryl or heteroaryl moieties. Q is asubstituted or unsubstituted methine moiety (e.g.,—(CH═C(R^(u)))_(c)—CH═), in which c is an integer selected from 1, 2, 3,4, or 5. Each R^(u), R^(w), R^(x), R^(y) and R^(z) is independentlyselected from various suitable substituents, and the indices w and z areindependently selected from the integers from 0 to 6.

In some embodiments, each R^(w) and R^(z) is independently a substitutedor unsubstituted alkyl, heteroalkyl, aryl, or heteroaryl group that iscoupled to the A-ring or B-ring either directly or through a carbonyl,amide, carbamide, ester, thioester, ether, thioether, or amino linkage.

In some embodiments, each R^(x) and R^(y), is independently an alkyl orheteroalkyl group, optionally substituted with a sulfonic acid,carboxylic acid, phosphonic acid, or phosphoric acid.

In some embodiments, each R^(u) is independently hydrogen, alkyl, orheteroalkyl.

Specific embodiments are described more thoroughly in the above-listedpatent publications. Among the dyes usefully included in the dye-labeledcompounds of the instant disclosure are the dyes shown in Table 1.

TABLE 1 Exemplary fluorescent dyes.

The shield element of the instant dye-labeled compounds may be any ofthe shield elements described above in the context of the nucleotidecompounds, without limitation. Shield elements are also described inU.S. Patent Application Publication Nos. 2015/0050659 A1 and2016/0237279 A1.

In some dye-labeled compound embodiments, the shield element decreasesphotodamage of the dye-labeled compound or of a biomolecule associatedwith the dye-labeled compound. In some compound embodiments, the shieldelement increases the brightness of the dye-labeled compound.

In specific compound embodiments, the shield element comprises aplurality of side chains. In some embodiments, at least one side chainhas a molecular weight of at least 300. In other embodiments, all of theside chains have a molecular weight of at least 300. In someembodiments, at least one side chain comprises a polyethylene glycol. Insome embodiments, at least one side chain comprises a negatively-chargedcomponent. More specifically, the negatively-charged component maycomprise a sulfonic acid. In some embodiments, at least one side chaincomprises a substituted phenyl group, more specifically the structure:

wherein each x is independently an integer from 1 to 6. Even morespecifically, each x may independently be an integer from 1 to 4. Insome embodiments, at least one side chain comprises a triazole, and insome embodiments at least one side chain may comprise a structureillustrated in FIG. 21.

In some dye-labeled compound embodiments, the shield element comprisesthe structure:

wherein each y is independently an integer from 1 to 6.

In other embodiments, the shield element comprises a structureillustrated in FIG. 21.

The shield elements of the instant dye-labeled compounds can in additionor alternatively comprise a dendrimer structure, including any of thedendrimer structures described above in the context of the nucleotidecompounds. An example of an intermediate compound used to generate adendrimer-containing dye-labeled compound of the instant disclosure isillustrated in FIG. 22.

This structure comprises two of the above-described G3 dendrimeric sidechains and four donor fluorophores with their associated shieldelements. It represents a higher-branched variant of the intermediatecompound shown in the left panel of FIG. 7G.

The instant dye-labeled compounds still further comprise a dye compoundlinker element. The dye compound linker element can be any of thelinkers defined above, as would be understood by those of ordinary skillin the art. The dye compound linker element serves to covalently connectthe terminal coupling element or elements with the donor dye or dyes,the acceptor dye or dyes, and the shield element or elements. In somecompound embodiments, more than one dye compound linker element may benecessary to connect the different components, as will be understood bythe skilled artisan upon consideration of the dye labeled compoundsexemplified below.

In some embodiments, the dye compound linker element comprises thestructure:

wherein each z is independently an integer from 1 to 8. In more specificembodiments, each z is independently an integer from 1 to 4. As isapparent in some of the compound examples described herein, the dyecompound linker element can further comprise an aminoalkyl group or adiaminoalkyl group. The dye compound linker element can alternatively oradditionally comprise other linker groups, for example, acylalkylgroups, diacylalkyl groups, or any other suitable linker group,including the branching groups described in U.S. Patent ApplicationPublication Nos. 2015/0050659 A1 and 2016/0237279 A1, and themultivalent central core elements described above. In some compoundembodiments, two or more dye compound linker elements are covalentlycoupled to one another.

In specific embodiments, the dye compound linker element comprises thestructure:

and in some embodiments comprises the structure

In some embodiments, the dye compound linker element comprises thestructure

Some dye compound linker elements can contain more than one of the abovestructures, and different dye compound linker element structures can bepresent within a single molecule of the instant compounds.

The dye-labeled compounds still further comprise a terminal couplingelement. It should be understood that the terminal coupling elements canbe any of the terminal coupling elements described above in the contextof the nucleotide compounds, without limitation. In some embodiments,the compounds comprise two terminal coupling elements. In someembodiments, the terminal coupling element comprises a biotin. Inpreferred embodiments, the terminal coupling element comprises abis-biotin, and in particular, one of the bis-biotin structures shownabove.

Exemplary dye-labeled compounds comprising a bis-biotin terminalcoupling element, at least one acceptor dye, at least one donor dye, andat least one dye compound linker element include the compoundillustrated in FIG. 23A, which includes one unshielded donor dye and oneunshielded acceptor dye; the compound illustrated in FIG. 23B, whichincludes two unshielded donor dyes and one unshielded acceptor dye; thecompound illustrated in FIG. 23C, which includes two unshielded dyes andtwo unshielded acceptor dyes; the compound illustrated in FIG. 23D,which includes two unshielded donor dyes and one shielded acceptor dye;the compound illustrated in FIG. 23E, which includes two shielded donordyes and one unshielded acceptor dye; the compound illustrated in FIG.23F, which includes two shielded donor dyes and two unshielded acceptordyes; the compound illustrated in FIG. 23G, which includes twounshielded donor dyes and two shielded acceptor dyes; the compoundillustrated in FIG. 23H, which includes two shielded donor dyes and oneshielded acceptor dye; the compound illustrated in FIG. 23I, whichincludes two shielded donor dyes and one shielded acceptor dye; and thecompound illustrated in FIG. 23J, which includes two shielded donor dyesand two shielded acceptor dyes.

Other exemplary dye-labeled compounds are illustrated as components ofthe labeled nucleotide analogs shown in FIGS. 3A-3Z and 3A′-3O′ andFIGS. 7A-7D, 7F, and 7G, and in the compounds graphically illustrated inFIGS. 4A-4C and FIGS. 5A-5M.

In preferred embodiments, the dye-labeled compounds of the instantdisclosure do not contain a polyphosphate element or a nucleosideelement.

As described above in the context of the instant nucleotide compounds,the terminal coupling element of the instant dye-labeled compoundstypically mediates association of the dye-labeled compound with othercomponents of the instant labeled nucleotide analogs. For example, andhas been described elsewhere in the disclosure, where the terminalcoupling element is a biotin or bis-biotin, the dye-labeled compound canassociate non-covalently with an avidin protein with high affinity. Insome aspects, the disclosure thus further provides compositionscomprising a dye-labeled compound of the disclosure and an avidinprotein. In these compositions, it should be understood that theterminal coupling element is not covalently modified by the associationof the dye-labeled compound with the avidin protein, and the compositionthus distinctly comprises the original dye-labeled compound and theavidin protein as separate molecular entities.

In another aspect of the disclosure, however, it should be contemplatedthat the terminal coupling element of a dye-labeled compound maycomprise a reactive functional group that can be covalently bound to acomplementary reactive group on a second component, for example on anappropriately modified linker element, shield element, or nucleotidecompound. Unlike the just-described non-covalent compositions, suchreactions generate a new molecular entity connected by a residue derivedfrom the reactive group of each component. As described elsewhere in thespecification, such residues can comprise, for example, an amide moietyderived from an amine group and an appropriately activated carboxylgroup or a residue resulting from a click reaction.

In yet another aspect of the disclosure are provided methods ofsynthesis of the instant dye-labeled compounds and their intermediates.Such methods can comprise the step of reacting any of the intermediatecompounds illustrated throughout the specification with a secondintermediate compound to generate a nucleotide compound or intermediateof the invention. Exemplary synthetic pathways are illustrated in thereaction schemes below, in the Examples, and in the accompanyingdrawings.

Synthesis and Assembly of Nucleotide and Dye-Labeled Compounds andAnalogs

In another aspect, the disclosure provides methods of synthesis andassembly of the compounds and labeled nucleotide analogs disclosedherein. These compounds and analogs are readily prepared using standardchemical techniques. Detailed examples of synthetic reactions that canbe adapted to prepare the instant compounds are provided in U.S. PatentApplication Publication Nos. 2015/0050659 A1 and 2016/0237279 A1. Forexample, the central core of exemplary shield elements can besynthesized according to the reactions illustrated in Scheme 1:

Core components of the shield element side chains can be synthesized,for example, according to the reactions illustrated in Scheme 2:

Core components of the shield element side chains can be synthesized,for example, according to the reactions illustrated in Scheme 2:

Shield elements modified with a nucleoside hexaphosphate can besynthesized, for example according to Schemes 3-1 or 3-2:

As would be understood from the above description, the shield elementswithin the final structures shown in Schemes 3-1 and 3-2 represent“layered” shield elements.

The shield core element reagent, TFA-Sh-CONHS, used in the initial stepof the first two reaction cycles of Scheme 3-1, can be generated byreaction of the “Sh” shield core element of Scheme 1 with TFA-NHS toform the following structure:

SG1-N₃ has the structure:

PEG7-N3 has the structure:

N3-Aba-CONHS has the structure:

NH₂-14C-dN6P represents a hexaphosphate deoxynucleotide containing a14-carbon, or equivalent, linker chain terminating in an amino group. Anexemplary species of this structure is:

wherein the nucleobase is thymine, and the C14-linker chain includes anamide bond.

Alternative pathways for generating shield element-containing reagentsuseful in the synthesis of various compounds of the instant disclosureare outlined in Schemes 4-1 to 4-3:

The shield elements prepared according to the above schemes correspondto “layered” shields, but the synthetic reactions can be suitablyaltered to generate non-layered shields if desired.

Exemplary synthetic reactions useful in the generation of theazide-containing sidechain reagents of Schemes 4-1 to 4-3 (e.g., R₁—N₃and R₂—N₃) are outlined in Scheme 5:

Reasonable variations in all of the above shield component intermediatestructures should be considered within the scope of the disclosure.

Exemplary synthetic schemes to generate other azide intermediates areillustrated in Scheme 6:

Exemplary reactions for preparing components of the just-describedshield elements are illustrated in Schemes 7-1 and 7-2:

An alternative reaction sequence for preparing components of variantshield elements of the instant nucleotide and dye-labeled compounds isillustrated in Scheme 8, in which the initial step is performed with asingle equivalent of the alkylating agent, thus resulting in theselective reaction at the 4-hydroxyl group.

Selective alkylation reactions can be used more generally to achieveincreased molecular diversity in the preparation of the instantcompounds, as is known in the art.

An exemplary synthetic route for preparing a dendrimer side chainsubstituent of the instant compounds and labeled nucleotide analogs isillustrated in FIG. 24.

In the reaction scheme of FIG. 24, further variability in structure canbe achieved through the use of the following exemplary alternativereagents in alternative versions of the illustrated reactions:

The generation of a dendrimer having bifunctional reactivity as a linkeris illustrated in the synthetic pathway of FIG. 25, where the productshown can be selectively deprotected by removal of the Boc group.

It should be generally understood that other coupling chemistry canprove suitable in synthesizing the compounds of the instant disclosure,as would be understood by those of skill in the art. Accordingly,reactions other than those exemplified in the synthetic schemes abovecan be utilized, without limitation.

An exemplary shielded dye-labeled intermediate compound usefullyincorporated into the dye-labeled compounds and analogs of the instantdisclosure is illustrated graphically in FIG. 6A. This particularintermediate has been, for example, used to generate the dye-labeledcompound illustrated in FIG. 5G and the labeled nucleotide analogillustrated in FIG. 3K. An exemplary chemical structure corresponding tothe illustration of FIG. 6A is provided in FIG. 6B, which comprises ashield element, including a shield core element that is directly coupledto the dye, a dye compound linker element intermediate comprising tworeactive azide groups, and another small side chain attached to the dyecompound linker element. In this exemplary intermediate compound, theazide groups can be coupled to other dye-labeled intermediate compoundsor to a terminal coupling element, such as a terminal coupling elementcomprising a bis-biotin, using “click” reactions, as will be illustratedbelow and in FIGS. 7A-7D, 7F, and 7G. The side chains of the shieldelement can be further varied, if desired. For example, the exemplarychemical structure of FIG. 6C has smaller side chains than the sidechains in the structure of FIG. 6B, whereas the exemplary chemicalstructure of FIG. 6D has larger side chains than the side chains in thestructure of FIG. 6B. The different sizes of the side chains in theseexamples arises from the inclusion of one or more side chain corestructures in the larger side chains in the structures of FIGS. 6B and6D. It should again be understood here that although the illustration ofFIG. 6A shows two large side chains and one small side chain, thuscorresponding to the chemical structure of FIG. 6B, the graphicillustrations provided in the disclosure should not be consideredlimiting in the size or exact locations of the components represented inthese illustrations.

FIG. 6E displays a synthetic scheme for another exemplary shieldeddye-labeled intermediate compound, this one containing four shieldeddonor dyes and a bis-biotin binding element. The final product is alsodisplayed in a graphic representation. Note that this intermediatecompound contains a cyclooctyne terminal group and is thus suitable forreaction with an azide-substituted component using a copper-free clickreaction. A variant exemplary intermediate compound containing fourshielded donor dyes and two azide terminal groups is illustrated in FIG.6F.

The above-described components, including nucleotide compounds,dye-labeled compounds, and the chemical intermediates used in thesynthesis of those compounds, can be used to assemble the labelednucleotide analogs of the instant disclosure, for example using thesteps outlined in FIGS. 7A-7D and 7F. As shown in FIG. 7A, exemplarylabeled nucleotide analogs comprising dG and dT can be prepared bystarting with a first dye-labeled intermediate compound comprising twoshielded donor dyes, a terminal coupling element (e.g., bis-biotin), anda dye compound linker element intermediate with a reactive terminalgroup. In the preparation of the exemplary dG nucleotide analog, thedye-labeled intermediate is first complexed with an avidin protein, asrepresented by the spherical structure. A second dye-labeledintermediate compound, this one containing two unshielded acceptor dyesconnected by a dye compound linker element intermediate with tworeactive terminal groups is next coupled to the partially assembledanalog. This coupling reaction is carried out with an excess of thecomplexed first dye-labeled intermediate and avidin, such that bothreactive terminal groups of the second dye-labeled intermediate compoundare modified by the reactive groups of two of the first intermediatedye-labeled compounds. The coupling reaction is preferably acopper-catalyzed or copper-free click reaction, but other suitablecoupling reactions could be employed to generate the intermediatecomplex. This complex, which comprises two avidin proteins and adye-labeled compound comprising two unshielded acceptor dyes, fourshielded donor dyes, three coupled dye compound linker elements, and twobis-biotin terminal coupling elements, is then reacted with an excess ofa dG nucleotide compound to generate the final dG analog product. Thenucleotide compounds used in all of the analogs shown in FIGS. 7A and 7Bcomprise a single nucleoside element (dG, dT, dA, or dC), apolyphosphate element, a nucleotide linker element comprising an anionicaromatic spacer element and a shield element, and a bis-biotin terminalcoupling element.

An exemplary dT nucleotide analog can be prepared, for example, by thepathway shown on the right side of FIG. 7A. According to this pathway,the first dye-labeled intermediate comprising two shielded donor dyes, aterminal coupling element (e.g., bis-biotin), and a dye compound linkerelement intermediate with a reactive terminal group is first coupled tothe second dye-labeled intermediate compound comprising two unshieldedacceptor dyes connected by a dye compound linker element intermediatewith two reactive terminal groups. An excess of the product of thecoupling reaction is complexed with an avidin protein to generate acomplex comprising one avidin protein and two of the partially coupleddye-labeled compound intermediates. This complex is next coupled to anexcess of the first avidin complex from the first pathway, whichcomprises an avidin protein and the dye-labeled complex intermediatewith two shielded donor dyes. As shown, the product of this couplingreaction comprises three avidin proteins and two of the dye-labeledcompounds described above for the dG analog. The dG and dT analogs canbe distinguished from one another by the difference in intensity offluorescence signal emitted from the each complex, because the dT analogcontains two dye-labeled compounds whereas the dG analog contains justone dye-labeled compound. Each of the dye-labeled compounds in the dGand dT analogs comprises four shielded donor dyes and two unshieldedacceptor dyes.

The dA and dC analogs can be assembled as outlined in the exemplarypathways of FIG. 7B. The primary difference between the pathways of FIG.7B and the pathways of FIG. 7A is the use of a first dye-labeledintermediate compound comprising two unshielded donor dyes. This firstintermediate is otherwise the same as the first dye-labeled intermediateof FIG. 7A which comprises two shielded donor dyes. The other differencein the pathways is the use of a second dye-labeled intermediate compoundcomprising two shielded acceptor dyes compared to the secondintermediate of FIG. 7A which comprises two unshielded acceptor dyes.The dA and dC analogs can be distinguished from one another by thedifference in intensity of fluorescence signal emitted from the eachcomplex, because the dC analog contains two dye-labeled compoundswhereas the dA analog contains just one dye-labeled compound. Each ofthe dye-labeled compounds in the dA and dC analogs comprises fourunshielded donor dyes and two shielded acceptor dyes. As with the dG anddT analogs, the dA and dC analogs can be distinguished from one anotherby the difference in intensity of fluorescence signal emitted from theeach complex. The dG analog is distinguishable from the dA analog andthe dT analog is distinguishable from the dC analog based on differencesin spectra of the different dye-labeled compounds due to the differentmicroenvironments of the shielded dyes.

FIGS. 7C and 7D illustrate alternative pathways useful in the assemblyof analogs comprising exemplary labeled dT, dG, dC, and dA nucleotideanalogs. FIG. 7E provides a legend for the relationship between some ofthe exemplary graphical illustrations of the figures and the chemicalstructures of the components represented in those illustrations. FIG. 7Fdisplays still other exemplary components and pathways that have beenused to prepare labeled nucleotide analogs of the instant disclosure.

Exemplary labeled nucleotide analogs of the instant disclosure thusinclude the specific analogs described in the following numberedparagraphs:

1. A labeled nucleotide analog comprising:

a first avidin protein having four subunits, each subunit comprising onebiotin binding site;

a first nucleotide compound bound to the first avidin protein, the firstnucleotide compound comprising a polyphosphate element, a nucleosideelement, an optional multivalent central core element, a terminalcoupling element, and a nucleotide linker element, wherein the firstnucleotide compound comprises at least one affinity modulating element;and

a first dye-labeled compound bound to the first avidin protein, thefirst dye-labeled compound comprising a donor dye, an acceptor dye, aterminal coupling element, and a dye compound linker element.

2. The labeled nucleotide analog of paragraph 1, wherein the firstdye-labeled compound and the first nucleotide compound are each bound tothe first avidin protein through a biotin moiety.

3. The labeled nucleotide analog of paragraph 1, further comprising asecond avidin protein and a second nucleotide compound, wherein

the second avidin protein is bound to the first dye-labeled compoundthrough a biotin moiety and to the second nucleotide compound through abiotin moiety.

4. The labeled nucleotide analog of paragraph 1, wherein the firstavidin protein is avidin, streptavidin, tamavidin, traptavidin,xenavidin, bradavidin, AVR2, AVR4, or a homolog thereof.

5. The labeled nucleotide analog of paragraph 1, wherein the donor dyeand the acceptor dye are fluorescent dyes.

6. The labeled nucleotide analog of paragraph 1, wherein the firstnucleotide compound is represented by structural formula (I):

wherein

L is the nucleotide linker element and comprises the affinity modulatingelement;

P is the polyphosphate element;

Nu is the nucleoside element;

X is the multivalent central core element;

B″ is the terminal coupling element and comprises a biotin moiety;

n is an integer from 1 to 4; and

o is 0 or 1.

7. The labeled nucleotide analog of paragraph 6, wherein the affinitymodulating element is an aromatic spacer element.

8. The labeled nucleotide analog of paragraph 7, wherein the aromaticspacer element is a substituted or unsubstituted monocyclic, bicyclic,tricyclic, or tetracyclic aromatic moiety.

9. The labeled nucleotide analog of paragraph 8, wherein the aromaticspacer element optionally comprises at least one anionic substituent.

10. The labeled nucleotide analog of paragraph 9, wherein the aromaticspacer element optionally comprises at least two anionic substituents.

11. The labeled nucleotide analog of paragraph 9, wherein the optionalat least one anionic substituent is —SO₃H.

12. The labeled nucleotide analog of paragraph 8, wherein the aromaticspacer element is represented by one of the following structuralformulae:

wherein

one of the A₁, A₂, A₃, and A₄ groups is

and the other groups are —CH₂— or a bond; and

R₁, R₂, R₃, R₄, R₅, and R₆ are independently H or an anionicsubstituent.

13. The labeled nucleotide analog of paragraph 12, wherein the aromaticspacer element is represented by one of the following structuralformulae:

14. The labeled nucleotide analog of paragraph 6, wherein L furthercomprises an alkyl linker group, optionally comprising an amide bond.15. The labeled nucleotide analog of paragraph 6, wherein L furthercomprises a triazole.16. The labeled nucleotide analog of paragraph 6, wherein B″ comprises abiotin moiety.17. The labeled nucleotide analog of paragraph 16, wherein B″ comprisesa bis-biotin moiety.18. The labeled nucleotide analog of paragraph 6, wherein o is 1.19. The labeled nucleotide analog of paragraph 18, wherein n is 2.20. The labeled nucleotide analog of paragraph 18, wherein X comprises apolyamine moiety.21. The labeled nucleotide analog of paragraph 6, wherein o is 0 and nis 1.22. The labeled nucleotide analog of paragraph 6, wherein the at leastone nucleotide compound does not contain a dye.23. The labeled nucleotide analog of any one of paragraphs 6-22, whereinL comprises at least one shield element.24. The labeled nucleotide analog of paragraph 23, wherein the shieldelement comprises a plurality of side chains.25. The labeled nucleotide analog of paragraph 24, wherein at least oneside chain has a molecular weight of at least 300.26. The labeled nucleotide analog of paragraph 24, wherein all of theside chains have a molecular weight of at least 300.27. The labeled nucleotide analog of paragraph 24, wherein at least oneside chain comprises a negatively-charged component.28. The labeled nucleotide analog of paragraph 27, wherein thenegatively-charged component comprises a sulfonic acid.29. The labeled nucleotide analog of paragraph 24, wherein at least oneside chain comprises a substituted phenyl group.30. The labeled nucleotide analog of paragraph 29, wherein the at leastone side chain comprises the structure:

wherein each x is independently an integer from 1 to 6.31. The labeled nucleotide analog of paragraph 30, wherein each x isindependently an integer from 1 to 4.32. The labeled nucleotide analog of paragraph 24, wherein at least oneside chain comprises a triazole.33. The labeled nucleotide analog of paragraph 24, wherein at least oneside chain comprises the structure:

34. The labeled nucleotide analog of paragraph 23, wherein the shieldelement comprises the structure:

wherein each y is independently an integer from 1 to 6.35. The labeled nucleotide analog of paragraph 23, wherein L comprisesthe structure:

36. The labeled nucleotide analog of paragraph 23, wherein L furthercomprises an alkyl linker group, optionally comprising an amide bond.37. The labeled nucleotide analog of paragraph 23, wherein L furthercomprises a triazole.38. The labeled nucleotide analog of paragraph 23, wherein B″ comprisesa biotin moiety.39. The labeled nucleotide analog of paragraph 38, wherein B″ comprisesa bis-biotin moiety.40. The labeled nucleotide analog of paragraph 23, wherein o is 1.41. The labeled nucleotide analog of paragraph 40, wherein n is 2.42. The labeled nucleotide analog of paragraph 40, wherein X comprises apolyamine moiety.43. The labeled nucleotide analog of paragraph 23, wherein o is 0 and nis 1.44. The labeled nucleotide analog of paragraph 23, wherein the at leastone nucleotide compound does not contain a dye.45. A labeled nucleotide analog comprising:

a first avidin protein, a second avidin protein, and a third avidinprotein, each avidin protein having four subunits, and each subunitcomprising one biotin binding site;

a first nucleotide compound and a second nucleotide compound, eachnucleotide compound comprising a polyphosphate element, a nucleosideelement, an optional multivalent central core element, a terminalcoupling element, and a nucleotide linker element, wherein the firstnucleotide compound or the second nucleotide compound comprises at leastone affinity modulating element; and

a first dye-labeled compound and a second dye-labeled compound, eachdye-labeled compound comprising a donor dye, an acceptor dye, a terminalcoupling element, and a dye compound linker element,

wherein the first avidin protein is bound to the first nucleotidecompound and to the first dye-labeled compound, the second avidinprotein is bound to the first dye-labeled compound and to the seconddye-labeled compound, and the third avidin protein is bound to thesecond dye-labeled compound and to the second nucleotide compound.

46. The labeled nucleotide analog of paragraph 45, wherein the first andsecond dye-labeled compounds and the first and second nucleotidecompounds are each bound to the first, second, and third avidin proteinsthrough biotin moieties.

Polymerase Enzymes

The labeled nucleotide analogs disclosed herein can be optimized andadapted for use with particular polymerase enzymes, in particularthrough structural modulation of the nucleotide compound components ofthe analogs. In addition, the polymerase enzymes can themselves beadapted for use with the analogs of the instant disclosure by directedmutation. In particular, a variety of natural and modified polymeraseenzymes are known in the art, and the structural and functionalproperties of these enzymes are well understood. DNA polymerases aresometimes classified into six main groups based upon variousphylogenetic relationships, e.g., with E. coli Pol I (class A), E. coliPol II (class B), E. coli Pol III (class C), Euryarchaeotic Pol II(class D), human Pol beta (class X), and E. coli UmuC/DinB andeukaryotic RAD30/xeroderma pigmentosum variant (class Y). For a reviewof nomenclature, see, e.g., Burgers et al. (2001) “Eukaryotic DNApolymerases: proposal for a revised nomenclature” J Biol Chem.276(47):43487-90. For a review of polymerases, see, e.g., Hübscher etal. (2002) “Eukaryotic DNA Polymerases” Annual Review of BiochemistryVol. 71: 133-163; Alba (2001) “Protein Family Review: Replicative DNAPolymerases” Genome Biology 2(1):reviews 3002.1-3002.4; and Steitz(1999) “DNA polymerases: structural diversity and common mechanisms” JBiol Chem 274:17395-17398. The basic mechanisms of action for manypolymerases have been determined. The sequences of hundreds ofpolymerases are publicly available, and the crystal structures for manyof these have been determined or can be inferred based upon similarityto solved crystal structures for homologous polymerases. For example,the crystal structure of Φ29, a preferred type of parental enzyme to bemodified according to the invention, is available. Many polymerases arecommercially available, e.g., for use in sequencing, labeling, andamplification technologies. Exemplary useful DNA polymerases include Taqand other thermostable polymerases, exonuclease deficient Taqpolymerases, E. coli DNA Polymerase I, Klenow fragment, reversetranscriptases, SP6 DNA polymerase, T7 DNA polymerase, T5 DNApolymerase, T4 DNA polymerase, RB69 polymerase, etc.

Enzymes particularly suitable for use with the analogs of the inventioninclude, but are not limited to, recombinant Φ29-type DNA polymerases. A“Φ29-type DNA polymerase” (or “phi29-type DNA polymerase”) is a DNApolymerase from the Φ29 phage or from one of the related phages that,like Φ29, contain a terminal protein used in the initiation of DNAreplication. Φ29-type DNA polymerases are homologous to the Φ29 DNApolymerase (e.g., as listed in SEQ ID NO:1); examples include the B103,GA-1, PZA, Φ15, BS32, M2Y (e.g., as listed in SEQ ID NO:2; also known asM2), Nf, G1, Cp-1, PRD1, PZE, SFS, Cp-5, Cp-7, PR4, PR5, PR722, L17,Φ21, and AV-1 DNA polymerases, as well as chimeras thereof. For example,the modified recombinant DNA polymerase can be homologous to a wild-typeor exonuclease deficient Φ29 DNA polymerase, e.g., as described in U.S.Pat. Nos. 5,001,050, 5,198,543, or 5,576,204. For nomenclature, seealso, Meijer et al. (2001) “Φ29 Family of Phages” Microbiology andMolecular Biology Reviews, 65(2):261-287. A modified recombinantΦ29-type DNA polymerase includes one or more mutations relative tonaturally-occurring wild-type Φ29-type DNA polymerases, for example, oneor more mutations that alter interaction with and/or incorporation ofnucleotide analogs, increase stability, increase readlength, enhanceaccuracy, increase phototolerance, and/or alter another polymeraseproperty, and can include additional alterations or modifications overthe wild-type Φ29-type DNA polymerase, such as one or more deletions,insertions, and/or fusions of additional peptide or protein sequences(e.g., for immobilizing the polymerase on a surface or otherwise taggingthe polymerase enzyme).

For example, a recombinant polymerase useful with analog(s) of theinvention can be homologous to (e.g., at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or even at least99% identical to) a wild type Φ29-type polymerase, e.g., to one of SEQID NOs:1-6. Amino acid residue identity is determined when the twosequences are compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Preferably, the identity exists over a region of the sequences that isat least about 50 residues in length, more preferably over a region ofat least about 100 residues, and most preferably, over at least about150 residues, or over the full length of the two sequences to becompared.

For reference, the amino acid sequence of a wild-type Φ29 polymerase ispresented in Table 2, along with the sequences of several otherwild-type Φ29-type polymerases.

TABLE 2 Amino acid sequence of exemplary wild-type Φ29-type polymerasesΦ29 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS SEQ IDEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWL NO: 1ERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK M2YMSRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKI SEQ IDGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEQH NO: 2GFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHTERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRKAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGVEPVELYLTNVDLELIQEHYELYNVEYIDGFKFREKTGLFKDFIDKWTYVKTHEEGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKDDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYVKEVDGKLKECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFAVGFSSMGKPKPVQVNGGVVLVDSVFTIK B103MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKI SEQ IDGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLEHH NO: 3GFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMFDSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK GA-1MARSVYVCDFETTTDPEDCRLWAWGWMDIYNTDKWS SEQ IDYGEDIDSFMEWALNSNSDIYFHNLKFDGSFILPWWLRN NO: 4GYVHTEEDRTNTPKEFTTTISGMGQWYAVDVCINTRGKNKNHVVFYDSLKKLPFKVEQIAKGFGLPVLKGDIDYKKYRPVGYVMDDNEIEYLKHDLLIVALALRSMFDNDFTSMTVGSDALNTYKEMLGVKQWEKYFPVLSLKVNSEIRKAYKGGFTWVNPKYQGETVYGGMVFDVNSMYPAMMKNKLLPYGEPVMFKGEYKKNVEYPLYIQQVRCFFELKKDKIPCIQIKGNARFGQNEYLSTSGDEYVDLYVTNVDWELIKKHYDIFEEEFIGGFMFKGFIGFFDEYIDRFMEIKNSPDSSAEQSLQAKLMLNSLYGKFATNPDITGKVPYLDENGVLKFRKGELKERDPVYTPMGCFITAYARENILSNAQKLYPRFIYADTDSIHVEGLGEVDAIKDVIDPKKLGYWDHEATFQRARYVRQKTYFIETTWKENDKGKLVVCEPQDATKVKPKIACAGMSDAIKERIRFNEFKIGYSTHGSLKPKNVLGGV VLMDYPFAIK AV-1MVRQSTIASPARGGVRRSHKKVPSFCADFETTTDEDDC SEQ IDRVWSWGIIQVGKLQNYVDGISLDGFMSHISERASHIYFH NO: 5NLAFDGTFILDWLLKHGYRWTKENPGVKEFTSLISRMGKYYSITVVFETGFRVEFRDSFKKLPMSVSAIAKAFNLHDQKLEIDYEKPRPIGYIPTEQEKRYQRNDVAIVAQALEVQFAEKMTKLTAGSDSLATYKKMTGKLFIRRFPILSPEIDTEIRKAYRGGFTYADPRYAKKLNGKGSVYDVNSLYPSVMRTALLPYGEPIYSEGAPRTNRPLYIASITFTAKLKPNHIPCIQIKKNLSFNPTQYLEEVKEPTTVVATNIDIELWKKHYDFKIYSWNGTFEFRGSHGFFDTYVDHFMEIKKNSTGGLRQIAKLHLNSLYGKFATNPDITGKHPTLKDNRVSLVMNEPETRDPVYTPMGVFITAYARKKTISAAQDNYETFAYADTDSLHLIGPTTPPDSLWVDPVELGAWKHESSFTKSVYIRAKQYAEEIGGKLDVHIAGMPRNVAATLTLEDMLHGGT WNGKLIPVRVPGGTVLKDTTFTLKID CP-1MTCYYAGDFETTTNEEETEVWLSCFAKVIDYDKLDTFK SEQ IDVNTSLEDFLKSLYLDLDKTYTETGEDEFIIFFHNLKFDGS NO: 6FLLSFFLNNDIECTYFINDMGVWYSITLEFPDFTLTFRDSLKILNFSIATMAGLFKMPIAKGTTPLLKHKPEVIKPEWIDYIHVDVAILARGIFAMYYEENFTKYTSASEALTEFKRIFRKSKRKFRDFFPILDEKVDDFCRKHIVGAGRLPTLKHRGRTLNQLIDIYDINSMYPATMLQNALPIGIPKRYKGKPKEIKEDHYYIYHIKADFDLKRGYLPTIQIKKKLDALRIGVRTSDYVTTSKNEVIDLYLTNFDLDLFLKHYDATIMYVETLEFQTESDLFDDYITTYRYKKENAQSPAEKQKAKIMLNSLYGKFGAKIISVKKLAYLDDKGILRFKNDDEEEVQPVYAPVALFVTSIARHFIISNAQENYDNFLYADTDSLHLFHSDSLVLDIDPSEFGKWAHEGRAVKAKYLRSKLYIEELIQEDGTTHLDVKGAGMTPEIKEKITFENFVIGATFEGKRASKQIK GGTLIYETTFKIRETDYLV

A recombinant polymerase useful with the analogs of the disclosure,e.g., a recombinant Φ29-type DNA polymerase, typically includes one ormore mutations (e.g., amino acid substitutions, deletions, orinsertions) as compared to a reference polymerase, e.g., a wild-typeΦ29-type polymerase, e.g., one of SEQ ID NOs:1-6. Depending on theparticular mutation or combination of mutations, the polymerase exhibitsone or more properties that find use in, e.g., single moleculesequencing applications or nucleic acid amplification. Such polymerasesincorporate nucleotides and/or nucleotide analogs, for example, theanalogs described herein, into a growing template copy during DNAamplification. These polymerases are modified such that they have one ormore desirable properties, for example, improved sequencing performancewith nucleotide analogs of the invention, increased readlength,increased thermostability, increased resistance to photodamage,decreased branching fraction formation when incorporating the relevantanalogs, improved DNA-polymerase complex stability or processivity,increased cosolvent resistance, reduced exonuclease activity, increasedyield, altered cofactor selectivity, improved accuracy, increased ordecreased speed, and/or altered kinetic properties (e.g., a reduction inthe rate of one or more steps of the polymerase kinetic cycle, resultingfrom, e.g., enhanced interaction of the polymerase with the nucleotideanalog, enhanced metal coordination, etc.) as compared to acorresponding wild-type or other parental polymerase (e.g., a polymerasefrom which the modified recombinant polymerase of the invention wasderived, e.g., by mutation).

Exemplary polymerases include a recombinant Φ29-type DNA polymerase thatcomprises a mutation (e.g., an amino acid substitution) at one or morepositions selected from the group consisting of S10, A68, A83, C106,A134, K135, L142, Y224, E239, V250, L253, A256, R261, R306, R308, L326,T368, T373, E375, E420, T421, T434, W436, A437, Y439, T441, C448, E466,D476, A484, 5487, E508, D510, K512, E515, K539, P558, D570, and T571,where identification of positions is relative to wild-type Φ29polymerase (SEQ ID NO:1). Optionally, the polymerase comprises mutationsat two or more, three or more, five or more, 10 or more, 15 or more, 20or more, or even 25 or more of these positions. A number of exemplarysubstitutions at these (and other) positions are described herein.Numbering of a given amino acid or nucleotide polymer “corresponds tonumbering of” or is “relative to” a selected amino acid polymer ornucleic acid when the position of any given polymer component (aminoacid residue, incorporated nucleotide, etc.) is designated by referenceto the same residue position in the selected amino acid or nucleotidepolymer, rather than by the actual position of the component in thegiven polymer. Similarly, identification of a given position within agiven amino acid or nucleotide polymer is “relative to” a selected aminoacid or nucleotide polymer when the position of any given polymercomponent (amino acid residue, incorporated nucleotide, etc.) isdesignated by reference to the residue name and position in the selectedamino acid or nucleotide polymer, rather than by the actual name andposition of the component in the given polymer. Correspondence ofpositions is typically determined by aligning the relevant amino acid orpolynucleotide sequences. For example, residue K221 of wild-type M2Ypolymerase (SEQ ID NO:2) is identified as position Y224 relative towild-type Φ29 polymerase (SEQ ID NO:1). Similarly, residue L138 ofwild-type M2Y polymerase (SEQ ID NO:2) is identified as position V141relative to wild-type Φ29 polymerase (SEQ ID NO:1), and an L138Ksubstitution in the M2Y polymerase is thus identified as a V141Ksubstitution relative to SEQ ID NO:1. Amino acid positions herein aregenerally identified relative to SEQ ID NO:1 unless explicitly indicatedotherwise.

As a few examples, a mutation at E375 can comprise an amino acidsubstitution selected from the group consisting of E375Y (i.e., atyrosine residue is present at position E375 where identification ofpositions is relative to SEQ ID NO:1), E375F, E375W, E375H, and E375M; amutation at position K512 can comprise an amino acid substitutionselected from the group consisting of K512Y, K512F, K512H, K512W, K512M,and K512R; a mutation at position L253 can comprise an L253Asubstitution; a mutation at position A484 can comprise an A484Esubstitution; and/or a mutation at position D510 can comprise a D510K orD510R substitution. Other exemplary substitutions include, e.g., S10M,A68S, A83E, C106S, A134S, K135Q, K135R, L142R, L142K, Y224K, E239G,V250I, A256S, R261K, R306Q, R308L, L326V, T368S, T373F, E420R, T421Y,T434A, T434S, W436Y, A437G, Y439W, T441I, C448V, E466K, D476H, S487A,E508R, E508Q, E515Q, K539E, P558A, D570S, and T571V; additionalsubstitutions are described herein.

The polymerase mutations noted herein can be combined with each otherand with essentially any other available mutations and mutationalstrategies to confer additional improvements in, e.g., nucleotide analogspecificity, enzyme processivity, improved retention time of labelednucleotides in polymerase-DNA-nucleotide complexes, phototolerance, andthe like. For example, the mutations and mutational strategies hereincan be combined with those taught in, e.g., U.S. Patent ApplicationPublication No. 2007/0196846; U.S. Patent Application Publication No.2008/0108082, U.S. Patent Application Publication No. 2010/0075332, U.S.Patent Application Publication No. 2010/0093555, U.S. Patent ApplicationPublication No. 2010/0112645, U.S. Patent Application Publication No.2011/0189659, U.S. Patent Application Publication No. 2012/0034602, U.S.Patent Application Publication 2013/0217007, U.S. Patent ApplicationPublication No. 2014/0094374, and U.S. Patent Application PublicationNo. 2014/0094375. Each of these applications is incorporated herein byreference in its entirety for all purposes. This combination ofmutations/mutational strategies can be used to impart severalsimultaneous improvements to a polymerase (e.g., enhanced utility withdesired analogs, increased readlength, increased phototolerance,decreased branching fraction formation, improved specificity, improvedprocessivity, altered rates, improved retention time, improved stabilityof the closed complex, tolerance for a particular metal cofactor, etc.).In addition, polymerases can be further modified forapplication-specific reasons, such as to improve activity of the enzymewhen bound to a surface, as taught, e.g., in U.S. Patent ApplicationPublication No. 2010/0261247 and U.S. Patent Application Publication No.2010/0260465 (each of which is incorporated herein by reference in itsentirety for all purposes) and/or to include purification or handlingtags as is taught in the cited references and as is common in the art.The polymerases can include one or more exogenous or heterologousfeatures, e.g., at the N-terminal region of the polymerase, at theC-terminal region of the polymerase, and/or internal to the polymerase.Such features find use not only for purification of the recombinantpolymerase and/or immobilization of the polymerase to a substrate, butcan also alter one or more properties of the polymerase. For additionalinformation on incorporation of such features, see, e.g., U.S. PatentApplication Publication Nos. 2012/0034602 and 2014/0094375 (each ofwhich is incorporated herein by reference in its entirety for allpurposes). Similarly, the modified polymerases described herein can beemployed in combination with other strategies to improve polymeraseperformance, for example, reaction conditions for controlling polymeraserate constants such as taught in U.S. Patent Application Publication No.2009/0286245, incorporated herein by reference in its entirety for allpurposes.

As noted, the various mutations described herein can be combined inrecombinant polymerases useful in the invention. Combination ofmutations can be random, or more desirably, guided by the properties ofthe particular mutations and the characteristics desired for theresulting polymerase. Additional mutations can also be introduced into apolymerase to compensate for deleterious effects of otherwise desirablemutations. For example, a W436Y substitution can reduce branchingfraction but induce pausing, Y439W can reduce pausing but also reduceyield, and R261K can increase yield; thus, a W436Y/Y439W/R261Kcombination can be desirable.

A number of exemplary mutations and the properties they confer aredescribed herein, and it will be evident that these mutations can befavorably combined in many different combinations. Exemplarycombinations are also provided herein, e.g., in Table 3, and an exampleof strategies by which additional favorable combinations are readilyderived follows. For the sake of simplicity, a few exemplarycombinations using only a few exemplary mutations are discussed, but itwill be evident that any of the mutations described herein can beemployed in such strategies to produce polymerases with desirableproperties.

For example, where a recombinant polymerase is desired to incorporateanalogs of the invention, one or more substitutions that enhance analogbinding through interactions with an aromatic group on the terminalphosphate, with a charged substituent on the aromatic group, and/or witha substituent elsewhere on the analog can be incorporated, e.g., anamino acid substitution at position K135, L142, T373, E375, and/or K512,e.g., K135Q, K135R, L142R, L142K, T373F, E375Y, E375F, E375W, E375H,E375M, D510R, K512Y, K512F, K512H, K512W, K512M, and/or K512R. As shownin FIG. 8, the tyrosine residues in a polymerase including E375Y andK512Y substitutions are positioned to stack with the DISC group on aDISC-containing hexaphosphate analog. In addition, the lysine atposition 135 forms a salt bridge with the DISC sulfonate group. As shownin FIG. 9, in a polymerase including E375W, K512F, and L142Rsubstitutions, the tryptophan and phenylalanine rings are positioned tostack with the DISC group, while the arginine at position 142 can form asalt bridge with an SG1 group elsewhere on the analog. As shown in FIG.10, in a polymerase including E375W, K512H, and K135R substitutions, thetryptophan and histidine rings are again positioned to interact with theDISC rings, and the arginine at position 135 forms a salt bridge withthe DISC sulfonate group. As shown in FIG. 11A, in a polymeraseincluding E375Y, K512Y, and D510R substitutions, the tyrosine residuescan stack with a 4,8-disulfonaphthalene-2,6-dicarboxylic acid (“DSDC”)spacer group.

The arginine at position 510 can form a salt bridge with one of the DSDCsulfonate groups, and the tyrosine at position 375 can hydrogen bond tothis sulfonate. The lysine at position 135 can form a salt bridge withthe other DSDC sulfonate group, which can also form a hydrogen bond withthe tyrosine at position 512. Similarly, as shown in FIG. 11B, in apolymerase including E375Y, K512Y, D510R, and K135R substitutions, thetyrosine residues can stack with the DSDC group. The arginine atposition 510 can form a salt bridge with one of the DSDC sulfonategroups, which can also form a hydrogen bond with the tyrosine atposition 375. The arginine at position 135 can form a bifurcated saltbridge with the other DSDC sulfonate group, which can also form ahydrogen bond with the tyrosine at position 512. Other substitutionsthat enhance analog binding (e.g., A484E) can also be incorporated intothe polymerase.

Where the polymerase is desired to incorporate the analogs in aMg⁺⁺-containing single molecule sequencing reaction, one or moresubstitutions that alter metal cofactor usage (e.g., L253A, L253H,L253C, or L253S) can be incorporated. Polymerase speed can be enhancedby inclusion of substitutions such as A437G, E508R, E508K, L142K, D510R,D510K, and/or V250I. Accuracy can be enhanced by inclusion ofsubstitutions such as E515Q and/or A134S. Processivity can be increasedby inclusion of substitutions such as D570S and/or T571V. Stabilityand/or yield can be increased by inclusion of substitutions such asY224K, E239G, and/or V250I. Stability can also be increased, e.g., byemploying M2Y as the parental polymerase and/or including astability-enhancing exogenous feature (e.g., a C-terminal exogenousfeature, e.g., a His10 or other polyhistidine tag). Use of largeanalogs, for example, analogs including protein moieties, canundesirably narrow pulse width and increase interpulse distance, so oneor more substitutions that increase pulse width (e.g., P558A, A256Sand/or S487A) or that decrease interpulse distance or reduce pausing(e.g., L142K, R306Q, R308L, T441I, C448V, E466K, D476H, and/or E508R)can be included in the polymerase. For discussion of pulse width andinterpulse distance, see, e.g., U.S. Patent Application Publication No.2014/0094375 (previously incorporated by reference in its entirety forall purposes).

It will be evident that different polymerase properties, and thereforedifferent combinations of mutations, are desirable for differentapplications involving recombinant polymerases. As will be understood, apolymerase can display one of the aforementioned properties alone or candisplay two or more of the properties in combination. Moreover, it willbe understood that while a particular mutation or polymerase can bedescribed with respect to a particular property, the mutation orpolymerase can possess additional modified properties not mentioned inevery instance for ease of discussion. It will also be understood thatparticular properties are observed under certain conditions. Forexample, a stability-improving mutation can, e.g., confer increasedstability on the polymerase-DNA substrate binary complex (as compared tosuch a complex containing a parental polymerase lacking the mutation)when observed in a thermal inactivation assay or it can confer increasedreadlength when observed in a single molecule sequencing reaction wherethe lifetime of the parental polymerase-DNA substrate complex (andtherefore readlength) is limited by its stability. A single mutation(e.g., a single amino acid substitution, deletion, insertion, or thelike) can give rise to one or more altered properties, or the one ormore properties can result from two or more mutations which act inconcert to confer the desired activity.

A list of exemplary mutations and combinations thereof is provided inTable 3, and additional exemplary mutations are described herein.Essentially any of these mutations, or any combination thereof, can beintroduced into a polymerase to produce a modified recombinantpolymerase (e.g., into wild-type Φ29 polymerase, wild-type M2polymerase, an exonuclease deficient Φ29 polymerase, or an exonucleasedeficient M2 polymerase, as just a few examples).

TABLE 3 Exemplary mutations introduced into a Φ29 DNA polymerase.Positions are identified relative to SEQ ID NO: 1. A685 C106S K135QL142R Y224K E239G V250I L253A R306Q R308L T368S E375W T421Y A437G E466KD476H A484E E508R D510R K512F E515Q K539E P558A D570S T571V A68S K135RL142K Y224K E239G V250I L253A R261K R306Q R308L L326V T368S E375W T421YW436Y A437G Y439W T441I C448V E466K D476H A484E E508Q D510R K512H E515QK539E P558A D570S T571V A68S C106S A134S K135R L142K Y224K E239G V250IL253A R261K R306Q R308L L326V E375F T421Y W436Y A437G Y439W E466K D476HA484E E508R D510R K512F E515Q K539E P558A D570S T571V A68S L142K Y224KE239G V250I L253A R306Q R308L T368S E375W T421Y A437G E466K D476H A484EE508R D510K K512F E515Q K539E P558A D570S T571V A68S C106S K135Q L142KY224K E239G V250I L253A R261K R306Q R308L L326V E375W T421Y W436Y A437GY439W E466K D476H A484E E508R D510R K512Y E515Q K539E P558A D570S T571VA68S K135R L142K Y224K E239G V250I L253A R261K R306Q R308L L326V E375WT421Y W436Y A437G Y439W E466K A484E E508R D510R K512H E515Q K539E P558AD570S T571V A68S C106S K135Q L142K Y224K E239G V250I L253A R261K R306QR308L L326V T373F E375Y T421Y W436Y A437G Y439W E466K D476H A484E E508RD510R K512Y E515Q K539E P558A D570S T571V A68S K135Q L142K Y224K E239GV250I L253A R261K R306Q R308L L326V E375Y T421Y W436Y A437G Y439W E466KD476H A484E E508R D510R K512Y E515Q K539E P558A D570S T571V A68S K135QL142K Y224K E239G V250I L253A R306Q R308L T368S E375Y T421Y A437G E466KD476H A484E E508R D510R K512Y E515Q K539E P558A D570S T571V A68S K135QL142K Y224K E239G V250I L253A R306Q R308L T368S E375Y T421Y A437G E466KD476H A484E E508R D510R K512Y E515Q K539E P558A D570S T571V

The amino acid sequences of exemplary recombinant Φ29 polymerasesharboring the exemplary mutation combinations of Table 3 are provided inTables 4 and 5. Table 4 includes the polymerase portion of the moleculeas well as one or more exogenous features at the C-terminal region ofthe polymerase, while Table 5 includes the amino acid sequence of thepolymerase portion only.

TABLE 4 Amino acid sequences of exemplary recombinantΦ29 polymerases including C-terminal exogenousfeatures. Amino acid positions are identified relative to SEQ ID NO: 1.Amino Acid Sequence SEQ ID NO: 7 MKHMPRKMYSCDFETTTKVEDCRVWAYA68S_C106S_K135Q_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ L142R_Y224K_E239G_ADLYFHNLKFDGSFIINWLERNGFKWSAD V250I_L253A_R306Q_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R308L_T368S_E375W_IHTVIYDSLKKLPFPVKKIAQDFKLTVRKG T421Y_A437G_E466K_DIDYHKERPVGYKITPEEYAYIKNDIQIIAE D476H_A484E_E508R_ALLIQFKQGLDRMTAGSDSLKGFKDIITTK D510R_K512F_E515Q_KFKKVFPTLSLGLDKEVRKAYRGGFTWLN K539E_P558A_D570S_DRFKGKEIGEGMVFDINSAYPAQMYSRLL T571V.co.GGGS.LVPPYGEPIVFEGKYVWDEDYPLHIQHIRCEFE RGS.GGGSGGGSGGGLKEGYIPTIQIKQSLFYKGNEYLKSSGGEIA S.BtagV7co.GGGSGGGDLWLSNVDLELMKEHYDLYNVEYISGLKF SGGGS.BtagV7co.G.KATTGLFKDFIDKWSYIKTTSWGAIKQLAK His10co LMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAWGRYTTI TAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQ DIYMKRVRGFLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQ VPGGVVLVDSVFTIKGGGSLVPRGSGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGG GSGGGSGLNDFFEAQKIEWHEGHHHHHH HHHHSEQ ID NO: 8 MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135R_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_T368S_KIHTVIYDSLKKLPFPVKKIARDFKLTVKK E375W_T421Y_W436Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A437G_Y439W_T441I_EALLIQFKQGLDRMTAGSDSLKGFKDIITT C448V_E466K_D476H_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL A484E_E508Q_D510R_NDRFKGKEIGEGMVFDINSAYPAQMYSKL K512H_E515Q_K539E_LPYGEPIVFEGKYVWDEDYPLHIQHIRCEF P558A_D570s_T571ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI V.co.G.His10co.GGADVWLSNVDLELMKEHYDLYNVEYISGL GSGGGSGGGS.BtagVKFKATTGLFKDFIDKWSYIKTTSWGAIKQL 7co.GGGSGGGSGGGSAKLMLNSLYGKFASNPDVTGKVPYLKENG .BtagV7co ALGFRLGEEEYKDPVYTPMGVFITAYGRWTIITAAQAVYDRIIYCDTDSIHLTGTKIPDV IKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKQVRGHLVQGSPDDYTDIKFSVK CAGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHHHHH GGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE SEQ ID NO: 9 MKHMPRKMYSCDFETTTKVEDCRVWAYA6SS_C106S_A134S_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ K135R_L142K_Y224K_ADLYFHNLKFDGSFIINWLERNGFKWSAD E239G_V250I_L253A_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R261K_R306Q_R308L_IHTVIYDSLKKLPFPVKKISRDFKLTVKKGD L326V_E375F_T421Y_IDYHKERPVGYKITPEEYAYIKNDIQIIAEA W436Y_A437G_Y439W_LLIQFKQGLDRMTAGSDSLKGFKDIITTKK E466K_D476H_A484E_FKKVFPTLSLGLDKEVRKAYRGGFTWLND E508R_D510R_K512F_RFKGKEIGEGMVFDINSAYPAQMYSKLLP E515Q_K539E_P558A_YGEPIVFEGKYVWDEDYPLHIQHIRCEFEL D570S_T571V.co.GKEGYIPTIQIKQSLFYKGNEYLKSSGGEIAD GGS.LVPRGS.GGGSGVWLSNVDLELMKEHYDLYNVEYISGLKFK GGSGGGS.BtagV7co.GATTGLFKDFIDKWTYIKTTSFGAIKQLAKL GGSGGGSGGGS.BtagMLNSLYGKFASNPDVTGKVPYLKENGALG V7co.G.His10coFRLGEEEYKDPVYTPMGVFITAYGRWTTIT AAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQD IYMKRVRGFLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQV PGGVVLVDSVFTIKGGGSLVPRGSGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGG SGGGSGLNDFFEAQKIEWHEGHHHHHHH HHHSEQ ID NO: 10 MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_L142K_Y224K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ E239G_V250I_L253A_ADLYFHNLKFDGSFIINWLERNGFKWSAD R306Q_R308L_T368S_GLPNTYNTIISRMGQWYMIDICLGYKGKR E375W_T421Y_A437G_KIHTVIYDSLKKLPFPVKKIAKDFKLTVKK E466K_D476H_A484E_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA E508R_D510K_K512F_EALLIQFKQGLDRMTAGSDSLKGFKDIITT E515Q_K539E_P558A_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL D570S_T571V.co.GNDRFKGKEIGEGMVFDINSAYPAQMYSRL GGSGGGSGGGS.BtagLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF V7co.GGGSGGGSGGGELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI S.BtagV7co.G.ADLWLSNVDLELMKEHYDLYNVEYISGLK His10co FKATTGLFKDFIDKWSYIKTTSWGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGA LGFRLGEEEYKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVI KDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGFLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGGGSGGGSGGGSG LNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHEGHHHHHHHHHH SEQ ID NO: 11 MKHMPRKMYSCDFETTTKVEDCRVWAYA68S_C106S_K135Q_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ L142K_Y224K_E239G_ADLYFHNLKFDGSFIINWLERNGFKWSAD V250I_L253A_R261K_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R306Q_R308L_L326V_IHTVIYDSLKKLPFPVKKIAQDFKLTVKKG E375W_T421Y_W436Y_DIDYHKERPVGYKITPEEYAYIKNDIQIIAE A437G_Y439W_E466K_ALLIQFKQGLDRMTAGSDSLKGFKDIITTK D476H_A484E_E508R_KFKKVFPTLSLGLDKEVRKAYRGGFTWLN D510R_K512Y_E515Q_DRFKGKEIGEGMVFDINSAYPAQMYSKLL K539E_P558A_D570S_PYGEPIVFEGKYVWDEDYPLHIQHIRCEFE T571V.co.GGGSGLKEGYIPTIQIKQSLFYKGNEYLKSSGGEIA GGSGGGS.BtagV7co.GDVWLSNVDLELMKEHYDLYNVEYISGLKF GGSGGGSGGGS.BtagKATTGLFKDFIDKWTYIKTTSWGAIKQLA V7co.G.His10coKLMLNSLYGKFASNPDVTGKVPYLKENGA LGFRLGEEEYKDPVYTPMGVFITAYGRWTTITAAQACYDRIIYCDTDSIHLTGTKIPDVI KDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGYLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGGGSGGGSGGGSG LNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHEGHHHHHHHHHH SEQ ID NO: 12 MKHMPRKMYSCDFETTTKVEDCRVWAYA68S_K135R_L142K_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_E375W_KIHTVIYDSLKKLPFPVKKIARDFKLTVKK T421Y_W436Y_A437G_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA Y439W_E466K_A484E_EALLIQFKQGLDRMTAGSDSLKGFKDIITT E508R_D510R_K512H_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL E515Q_K539E_P558A_NDRFKGKEIGEGMVFDINSAYPAQMYSKL D570S_T571V.coLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF .G.His10co.GGGSGGGELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI SGGGS.BtagV7co.GGGADVWLSNVDLELMKEHYDLYNVEYISGL SGGGSGGGS.BtagV7cKFKATTGLFKDFIDKWTYIKTTSWGAIKQL o (-D476H) AKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRW TTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTY IQDIYMKRVRGHLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAV QVPGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGG SGGGSGGGSGLNDFFEAQKIEWHE SEQ ID NO: 13MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_C106S_K135Q_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ L142K_Y224K_E239G_ADLYFHNLKFDGSFIINWLERNGFKWSAD V250I_L253A_R261K_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R306Q_R308L_L326V_IHTVIYDSLKKLPFPVKKIAQDFKLTVKKG T373F_E375Y_T421Y_DIDYHKERPVGYKITPEEYAYIKNDIQIIAE W436Y_A437G_Y439W_ALLIQFKQGLDRMTAGSDSLKGFKDIITTK E466K_D476H_A484E_KFKKVFPTLSLGLDKEVRKAYRGGFTWLN E508R_D510R_K512Y_DRFKGKEIGEGMVFDINSAYPAQMYSKLL E515Q_K539E_P558A_PYGEPIVFEGKYVWDEDYPLHIQHIRCEFE D570S_T571V.co.GLKEGYIPTIQIKQSLFYKGNEYLKSSGGEIA GGS.LVPRGS.GGGSGDVWLSNVDLELMKEHYDLYNVEYISGLKF GGSGGGS.BtagV7co.GKATTGLFKDFIDKWTYIKTFSYGAIKQLAK GGSGGGSGGGS.BtagLMLNSLYGKFASNPDVTGKVPYLKENGAL V7co.G.His10coGFRLGEEEYKDPVYTPMGVFITAYGRWTTI TAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQ DIYMKRVRGYLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQ VPGGVVLVDSVFTIKGGGSLVPRGSGGGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGG GSGGGSGLNDFFEAQKIEWHEGHHHHHH HHHHSEQ ID NO: 14 MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135Q_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_E375Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK T421Y_W436Y_A437G_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA Y439W_E466K_D476H_EALLIQFKQGLDRMTAGSDSLKGFKDIITT A484E_E508R_D510R_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL K512Y_E515Q_K539E_NDRFKGKEIGEGMVFDINSAYPAQMYSKL P558A_D570S_T571LPYGEPIVFEGKYVWDEDYPLHIQHIRCEF V.co.G.His10co.GGGELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI SGGGSGGGS.BtagV7cADVWLSNVDLELMKEHYDLYNVEYISGL o.GGGSGGGSGGGS.BKFKATTGLFKDFIDKWTYIKTTSYGAIKQL tagV7co AKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRW TTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTY IQDIYMKRVRGYLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAV QVPGGVVLVDSVFTIKGHHHHHHHHHHGGGSGGGSGGGSGLNDFFEAQKIEWHEGGG SGGGSGGGSGLNDFFEAQKIEWHE SEQ ID NO: 15MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135Q_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R306Q_R308L_GLPNTYNTIISRMGQWYMIDICLGYKGKR T368S_E375Y_T421Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK A437G_E466K_D476H_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A484E_E508R_D510R_EALLIQFKQGLDRMTAGSDSLKGFKDIITT K512Y_E515Q_K539E_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL P558A_D570S_T571NDRFKGKEIGEGMVFDINSAYPAQMYSRL V.co.GGGSGGGSGGGLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF S.B tagV7co.GGGSGGELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI GSGGGS.BtagV7co.G.ADLWLSNVDLELMKEHYDLYNVEYISGLK His10co FKATTGLFKDFIDKWSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGA LGFRLGEEEYKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVI KDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGYLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGGGSGGGSGGGSG LNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHEGHHHHHHHHHH SEQ ID NO: 16 MKHMPRKMYSCDFETTTKVEDCRVWAYA68S_K135Q_L142K_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R306Q_R308L_GLPNTYNTIISRMGQWYMIDICLGYKGKR T368S_E375Y_T421Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK A437G_E466K_D476H_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A484E_E508R_D510R_EALLIQFKQGLDRMTAGSDSLKGFKDIITT K512Y_E515Q_K539E_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL P558A_D570S_T571V.NDRFKGKEIGEGMVFDINSAYPAQMYSRL co.G.His10co.GGGSLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF GGGSGGGS.BtagV7co.ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI GGGSGGGSGGGS.BtaADLWLSNVDLELMKEHYDLYNVEYISGLK gV7co FKATTGLFKDFIDKWSYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGA LGFRLGEEEYKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVI KDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGYLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIKGHHHHHHHHHHG GGSGGGSGGGSGLNDFFEAQKIEWHEGGGSGGGSGGGSGLNDFFEAQKIEWHE

TABLE 5 Amino acid sequences of exemplary recombinantΦ29 polymerases. Amino acid positions areidentified relative to SEQ ID NO: 1. Amino Acid Sequence SEQ ID NO: 17MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_C106S_K135Q_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ L142R_Y224K_E239G_ADLYFHNLKFDGSFIINWLERNGFKWSAD V250I_L253A_R306Q_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R308L_T368S_E375W_IHTVIYDSLKKLPFPVKKIAQDFKLTVRKG T421Y_A437G_E466K_DIDYHKERPVGYKITPEEYAYIKNDIQIIAE D476H_A484E_E508R_ALLIQFKQGLDRMTAGSDSLKGFKDIITTK D510R_K512F_E515Q_KFKKVFPTLSLGLDKEVRKAYRGGFTWLN K539E_P558A_D570S_DRFKGKEIGEGMVFDINSAYPAQMYSRLL T571V PYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIA DLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWSYIKTTSWGAIKQLAK LMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAWGRYTTI TAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQ DIYMKRVRGFLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQ VPGGVVLVDSVFTIK SEQ ID NO: 18MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135R_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_T368S_KIHTVIYDSLKKLPFPVKKIARDFKLTVKK E375W_T421Y_W436Y_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A437G_Y439W_T441I_EALLIQFKQGLDRMTAGSDSLKGFKDIITT C448V_E466K_D476H_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL A484E_E508Q_D510R_NDRFKGKEIGEGMVFDINSAYPAQMYSKL K512H_E515Q_K539E_LPYGEPIVFEGKYVWDEDYPLHIQHIRCEF P558A_D570S_T571VELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI ADVWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWSYIKTTSWGAIKQL AKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRW TIITAAQAVYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTY IQDIYMKQVRGHLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKA VQVPGGVVLVDSVFTIK SEQ ID NO: 19MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_C106S_A134S_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ K135R_L142K_Y224K_ADLYFHNLKFDGSFIINWLERNGFKWSAD E239G_V250I_L253A_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R261K_R306Q_R308L_IHTVIYDSLKKLPFPVKKISRDFKLTVKKGD L326V_E375F_T421Y_IDYHKERPVGYKITPEEYAYIKNDIQIIAEA W436Y_A437G_Y439W_LLIQFKQGLDRMTAGSDSLKGFKDIITTKK E466K_D476H_A484E_FKKVFPTLSLGLDKEVRKAYRGGFTWLND E508R_D510R_K512F_RFKGKEIGEGMVFDINSAYPAQMYSKLLP E515Q_K539E_P558A_YGEPIVFEGKYVWDEDYPLHIQHIRCEFEL D570S_T571VKEGYIPTIQIKQSLFYKGNEYLKSSGGEIAD VWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSFGAIKQLAKL MLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRWTTIT AAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQD IYMKRVRGFLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQV PGGVVLVDSVFTIK SEQ ID NO: 20MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_L142K_Y224K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ E239G_V250I_L253A_ADLYFHNLKFDGSFIINWLERNGFKWSAD R306Q_R308L_T368S_GLPNTYNTIISRMGQWYMIDICLGYKGKR E375W_T421Y_A437G_KIHTVIYDSLKKLPFPVKKIAKDFKLTVKK E466K_D476H_A484E_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA E508R_D510K_K512F_EALLIQFKQGLDRMTAGSDSLKGFKDIITT E515Q_K539E_P558A_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL D570S_T571V NDRFKGKEIGEGMVFDINSAYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLK FKATTGLFKDFIDKWSYIKTTSWGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGA LGFRLGEEEYKDPVYTPMGVFITAWGRYTTITAAQACYDRIIYCDTDSIHLTGTKIPDVI KDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVKGFLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIK SEQ ID NO: 21 mkhmprkmyscdfetttkvedcrvwaygymnA68S_C106S_K135Q_ iedhseykignsldefmawvlkvqadlyfhn L142K_Y224K_E239G_lkfdgsfiinwlerngfkwsadglpntynti V250I_L253A_R261K_isrmgqwymidislgykgkrkihtviydslk R306Q_R308L_L326V_klpfpvkkiaqdfkltvkkgdidyhkerpvg E375W_T421Y_W436Y_ykitpeeyayikndiqiiaealliqfkqgld A437G_Y439W_E466K_rmtagsdslkgfkdiittkkfkkvfptlslg D476H_A484E_E508R_ldkevrkayrggftwlndrfkgkeigegmvf D510R_K512Y_E515Q_dinsaypaqmyskllpygepivfegkyvwde K539E_P558A_D570S_dyplhiqhircefelkegyiptiqikqslfy T571V kgneylkssggeiadvwlsnvdlelmkehydlynveyisglkfkattglfkdfidkwtyikt tswgaikqlaklmlnslygkfasnpdvtgkvpylkengalgfrlgeeeykdpvytpmgvfit aygrwttitaaqacydriiycdtdsihltgtkipdvikdivhpkklgywehestfkrakylr qktyiqdiymkrvrgylvqgspddytdikfsvkcagmtdkikeevtfenfkvgfsrkmkpka vqvpggvvlvdsvftik SEQ ID NO: 22MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135R_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_E375W_KIHTVIYDSLKKLPFPVKKIARDFKLTVKK T421Y_W436Y_A437G_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA Y439W_E466K_A484E_EALLIQFKQGLDRMTAGSDSLKGFKDIITT E508R_D510R_K512H_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL E515Q_K539E_P558A_NDRFKGKEIGEGMVFDINSAYPAQMYSKL D570S_T571V LPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI ADVWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSWGAIKQL AKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRW TTITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVDPKKLGYWEHESTFKRAKYLRQKTY IQDIYMKRVRGHLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAV QVPGGVVLVDSVFTIK SEQ ID NO: 23MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_C106S_K135Q_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ L142K_Y224K_E239G_ADLYFHNLKFDGSFIINWLERNGFKWSAD V250I_L253A_R261K_GLPNTYNTIISRMGQWYMIDISLGYKGKRK R306Q_R308L_L326V_IHTVIYDSLKKLPFPVKKIAQDFKLTVKKG T373F_E375Y_T421Y_DIDYHKERPVGYKITPEEYAYIKNDIQIIAE W436Y-A437G_Y439W_ALLIQFKQGLDRMTAGSDSLKGFKDIITTK E466K_D476H_A484E_KFKKVFPTLSLGLDKEVRKAYRGGFTWLN E508R_D510R_K512Y_DRFKGKEIGEGMVFDINSAYPAQMYSKLL E515Q_K539E_P558A_PYGEPIVFEGKYVWDEDYPLHIQHIRCEFE D570S_T571VLKEGYIPTIQIKQSLFYKGNEYLKSSGGEIA DVWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTFSYGAIKQLAK LMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAYGRWTTI TAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQ DIYMKRVRGYLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAVQ VPGGVVLVDSVFTIK SEQ ID NO: 24MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135Q_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R261K_R306Q_GLPNTYNTIISRMGQWYMIDICLGYKGKR R308L_L326V_E375Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK T421Y_W436Y_A437G_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA Y439W_E466K_D476H_EALLIQFKQGLDRMTAGSDSLKGFKDIITT A484E_E508R_D510R_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL K512Y_E515Q_K539E_NDRFKGKEIGEGMVFDINSAYPAQMYSKL P558A_D570S_T571VLPYGEPIVFEGKYVWDEDYPLHIQHIRCEF ELKEGYIPTIQIKQSLFYKGNEYLKSSGGEIADVWLSNVDLELMKEHYDLYNVEYISGL KFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENG ALGFRLGEEEYKDPVYTPMGVFITAYGRWTTITAAQACYDRIIYCDTDSIHLTGTKIPDV IKDIVHPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKRVRGYLVQGSPDDYTDIKFSVKC AGMTDKIKEEVTFENFKVGFSRKMKPKAVQVPGGVVLVDSVFTIK SEQ ID NO: 25 MKHMPRKMYSCDFETTTKVEDCRVWAYA68S_K135Q_L142K_ GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R306Q_R308L_GLPNTYNTIISRMGQWYMIDICLGYKGKR T368S_E375Y_T421Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK A437G_E466K_D476H_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A484E_E508R_D510R_EALLIQFKQGLDRMTAGSDSLKGFKDIITT K512Y_E515Q_K539E_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL P558A_D570S_T571VNDRFKGKEIGEGMVFDINSAYPAQMYSRL LPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI ADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWSYIKTTSYGAIKQLA KLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAWGRYT TITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYI QDIYMKRVRGYLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAV QVPGGVVLVDSVFTIK SEQ ID NO: 26MKHMPRKMYSCDFETTTKVEDCRVWAY A68S_K135Q_L142K_GYMNIEDHSEYKIGNSLDEFMAWVLKVQ Y224K_E239G_V250I_ADLYFHNLKFDGSFIINWLERNGFKWSAD L253A_R306Q_R308L_GLPNTYNTIISRMGQWYMIDICLGYKGKR T368S_E375Y_T421Y_KIHTVIYDSLKKLPFPVKKIAQDFKLTVKK A437G_E466K_D476H_GDIDYHKERPVGYKITPEEYAYIKNDIQIIA A484E_E508R_D510R_EALLIQFKQGLDRMTAGSDSLKGFKDIITT K512Y_E515Q_K539E_KKFKKVFPTLSLGLDKEVRKAYRGGFTWL P558A_D570S_T571VNDRFKGKEIGEGMVFDINSAYPAQMYSRL LPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKQSLFYKGNEYLKSSGGEI ADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWSYIKTTSYGAIKQLA KLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEEYKDPVYTPMGVFITAWGRYT TITAAQACYDRIIYCDTDSIHLTGTKIPDVIKDIVHPKKLGYWEHESTFKRAKYLRQKTYI QDIYMKRVRGYLVQGSPDDYTDIKFSVKCAGMTDKIKEEVTFENFKVGFSRKMKPKAV QVPGGVVLVDSVFTIK

Compositions, kits, and systems (e.g., sequencing systems) includingsuch recombinant polymerases, e.g., in combination with one or more ofthe instant labeled nucleotide analogs, are features of the disclosure,as are methods employing the recombinant polymerases (e.g., methods ofsequencing or making DNA). Many other such recombinant polymerasesincluding these mutations and/or those described elsewhere herein willbe readily apparent and are features of the disclosure.

The structures of Φ29 polymerase, Φ29 polymerase complexed with terminalprotein, and Φ29 polymerase complexed with primer-template DNA in thepresence and absence of a nucleoside triphosphate are available; seeKamtekar et al. (2004) “Insights into strand displacement andprocessivity from the crystal structure of the protein-primed DNApolymerase of bacteriophage Φ29” Mol. Cell 16(4): 609-618), Kamtekar etal. (2006) “The phi29 DNA polymerase:protein-primer structure suggests amodel for the initiation to elongation transition” EMBO J.25(6):1335-43, and Berman et al. (2007) “Structures of phi29 DNApolymerase complexed with substrate: The mechanism of translocation inB-family polymerases” EMBO J. 26:3494-3505, respectively. The structuresof additional polymerases or complexes can be modeled, for example,based on homology of the polymerases with polymerases whose structureshave already been determined. Alternatively, the structure of a givenpolymerase (e.g., a wild-type or modified polymerase), optionallycomplexed with a DNA (e.g., template and/or primer) and/or nucleotideanalog, or the like, can be determined using techniques known in theart. See, e.g., U.S. Patent Application Publication No. 2014/0094375 andreferences therein.

Mutations can be introduced into a desired parental polymerase and theresulting recombinant polymerase can be expressed, purified, andcharacterized (e.g., to determine one or more properties, e.g., for ananalog of the invention) using techniques known in the art. See, e.g.,U.S. Patent Application Publication Nos. 2007/0196846, 2008/0108082,2010/0075332, 2010/0093555, 2010/0112645, 2011/0189659, 2012/0034602,2013/0217007, 2014/0094374, and 2014/0094375 (previously incorporated byreference in their entirety for all purposes), and references therein.

Reaction Mixtures, Methods, and Systems for Nucleic Acid Sequencing

The disclosure further provides, in another aspect, reaction mixturesuseful in the sequencing of nucleic acids. Such mixtures preferablycomprise a polymerase enzyme complex that includes a polymerase enzyme,a template nucleic acid, and optionally a primer hybridized to thetemplate nucleic acid. Such polymerase complexes are ideally configuredfor immobilization on a surface, such as the surface of a ZMW. Thereaction mixtures additionally comprise sequencing reagents in contactwith the surface to which the polymerase complex is immobilized. Thesequencing reagents include nucleotides for carrying out nucleic acidsynthesis, in particular two or more of the labeled nucleotide analogsdescribed in detail above. Further details relating to the reactionmixtures, including preferred template nucleic acids, polymeraseenzymes, methods for immobilizing polymerase complexes to a surface,reaction conditions, including buffers, pH, salts, and the like, areprovided, for example, in U.S. Patent Application Publication No.2013/0316912 A1. Exemplary mutated polymerase enzymes usefully includedin the instant reaction mixtures with analogs comprising the instantmodified nucleotide compounds are described above.

In specific embodiments, the labeled nucleotide analog of the reactionmixture comprises at least one dye-labeled compound and at least onenucleotide compound, wherein the at least one dye-labeled compound andthe at least one nucleotide compound are described above. In morespecific embodiments, each dye-labeled compound and each nucleotidecompound comprises a bis-biotin moiety.

Exemplary reaction mixtures of the disclosure suitable for sequencingnucleic acids thus include the mixtures described in the followingnumbered paragraphs:

1. A reaction mixture for sequencing a nucleic acid template comprising:

a polymerase enzyme complex comprising a polymerase enzyme, a templatenucleic acid, and optionally a primer hybridized to the template nucleicacid, wherein the polymerase enzyme complex is immobilized on a surface;and

sequencing reagents in contact with the surface comprising reagents forcarrying out nucleic acid synthesis including two or more of any of thelabeled nucleotide analogs described above.

2. The reaction mixture of paragraph 1, wherein the labeled nucleotideanalog comprises one dye-labeled compound and one nucleotide compound,wherein the dye-labeled compound and the nucleotide compound eachcomprises a biotin moiety.

The disclosure still further provides, in yet another aspect, methodsfor sequencing a nucleic acid template. In these methods, a polymeraseenzyme complex comprising a polymerase enzyme, a template nucleic acid,and optionally a primer hybridized to the template nucleic acid isprovided. In some embodiments, the polymerase enzyme complex isimmobilized on a surface. Sequencing reagents are added to thepolymerase enzyme complex, wherein the reagents include nucleotides forcarrying out nucleic acid synthesis, in particular two or more of thelabeled nucleotide analogs described in detail above. The sequentialaddition of nucleotides to a nucleic acid strand complementary to astrand of the template nucleic acid is determined by observing theinteraction of the labeled nucleotide analogs with the polymerase enzymecomplex.

In specific method embodiments, the labeled nucleotide analog of thesequencing method comprises at least one dye-labeled compound and atleast one nucleotide compound of the instant disclosure. In morespecific method embodiments, the at least one dye-labeled compound andthe at least one nucleotide compound each comprise a bis-biotin moiety.

For example, methods of the disclosure suitable for sequencing a nucleicacid template include any of the methods described in the followingnumbered paragraphs:

1. A method for sequencing a nucleic acid template comprising:

providing a polymerase enzyme complex comprising a polymerase enzyme, atemplate nucleic acid, and optionally a primer hybridized to thetemplate nucleic acid, wherein the polymerase enzyme complex isimmobilized on a surface;

adding sequencing reagents in contact with the surface comprisingreagents for carrying out nucleic acid synthesis including two or moreof any of the labeled nucleotide analogs described above; and

determining the sequential addition of nucleotides to a nucleic acidstrand complementary to a strand of the template nucleic acid byobserving the interaction of the labeled nucleotide analogs with thepolymerase enzyme complex.

2. The method of paragraph 1, wherein at least one labeled nucleotideanalog comprises:

one nucleotide compound bound to the avidin protein through a biotinmoiety; and

one dye-labeled compound bound to the avidin protein through a biotinmoiety.

3. The method of paragraph 1, wherein at least one labeled nucleotideanalog comprises:

one dye-labeled compound, bound to the avidin protein through a biotinmoiety; and

two nucleotide compounds, each bound to the avidin protein through abiotin moiety.

4. The method of paragraph 1, wherein at least one labeled nucleotideanalog comprises:

one dye-labeled compound and one nucleotide compound, each of which isbound to the avidin protein through a biotin moiety.

5. The method of paragraph 1, wherein at least one labeled nucleotideanalog comprises:

two avidin proteins, two nucleotide compounds, and one dye-labeledcompound; wherein

the dye-labeled compound comprises two biotin moieties;

each nucleotide compound comprises one biotin moiety; and

each avidin protein is bound to the dye-labeled compound through abiotin moiety and to one of the nucleotide compounds through a biotinmoiety.

6. The method of any one of paragraphs 1-5, wherein the avidin proteincomprises either streptavidin or tamavidin.

7. The method of any one of paragraphs 1-5, wherein the dye moieties arefluorescent dyes.

8. The method of any one of paragraphs 1-5, wherein at least onenucleotide compound is represented by structural formula (I):

wherein

L is a nucleotide linker element comprising an affinity modulatingelement;

P is a polyphosphate element;

Nu is a nucleoside element;

X is a multivalent central core element;

B″ is a terminal coupling element;

n is an integer from 1 to 4; and

o is 0 or 1.

9. The method of paragraph 8, wherein the affinity modulating element isan aromatic spacer element.

10. The method of paragraph 9, wherein the aromatic spacer element is asubstituted or unsubstituted monocyclic, bicyclic, tricyclic, ortetracyclic aromatic moiety.

11. The method of paragraph 10, wherein the aromatic spacer elementoptionally comprises at least one anionic substituent.

12. The method of paragraph 11, wherein the aromatic spacer elementoptionally comprises at least two anionic substituents.

13. The method of paragraph 11, wherein the optional at least oneanionic substituent is —SO₃H.

14. The method of paragraph 10, wherein the aromatic spacer element isrepresented by one of the following structural formulae:

wherein

one of the A₁, A₂, A₃, and A₄ groups is

and the other groups are —CH₂— or a bond; and

R₁, R₂, R₃, R₄, R₅, and R₆ are independently H or an anionicsubstituent.

15. The method of paragraph 14, wherein the aromatic spacer element isrepresented by one of the following structural formulae:

16. The method of paragraph 8, wherein L further comprises an alkyllinker group, optionally comprising an amide bond.17. The method of paragraph 8, wherein L further comprises a triazole.18. The method of paragraph 8, wherein B″ comprises a biotin moiety.19. The method of paragraph 18, wherein B″ comprises a bis-biotinmoiety.20. The method of paragraph 8, wherein o is 1.21. The method of paragraph 20, wherein n is 2.22. The method of paragraph 20, wherein X comprises a polyamine moiety.23. The method of paragraph 8, wherein o is 0 and n is 1.24. The method of any one of paragraphs 1-5, wherein at least onedye-labeled compound comprises:

a donor dye;

an acceptor dye;

a shield element;

a terminal coupling element; and

a dye compound linker element;

wherein the dye compound linker element covalently connects the terminalcoupling element to the donor dye, the acceptor dye, or the shieldelement; and wherein the acceptor dye or the donor dye is directlycoupled to the shield element.

25. The method of paragraph 24, wherein the terminal coupling elementcomprises a biotin moiety.

26. The method of paragraph 25, wherein the terminal coupling elementcomprises a bis-biotin moiety.

27. The method of paragraph 8, wherein o is 1.

28. The method of paragraph 27, wherein n is 2.

29. The method of paragraph 28, wherein X comprises a polyamine moiety.

30. The method of paragraph 24, wherein o is 0 and n is 1.

In yet another aspect, the disclosure provides systems for sequencingnucleic acids. Such systems preferably comprise a chip comprising aplurality of polymerase enzyme complexes bound thereto, each polymeraseenzyme complex individually optically resolvable, each polymerase enzymecomplex comprising a polymerase enzyme, a template nucleic acid, andoptionally a primer hybridized to the template nucleic acid. The systemfurther comprises sequencing reagents in contact with the surface. Thesequencing reagents comprise reagents for carrying out nucleic acidsynthesis, including two or more of the labeled nucleotide analogsdescribed in detail above. The system also comprises an illuminationsystem for illuminating the polymerase enzyme complexes, an opticaldetection system for detecting fluorescence from the labeled nucleotideanalogs while they are interacting with the polymerase enzyme complexes,and a computer for analyzing the signals detected by the detectionsystem to determine the sequential addition of nucleotides to a nucleicacid strand complementary to a strand of the template nucleic acid. Suchsystems are further described, for example, in U.S. Patent ApplicationPublication No. 2013/0316912 A1.

For example, the system for sequencing nucleic acids can comprise:

a chip comprising a plurality of polymerase enzyme complexes boundthereto, each polymerase enzyme complex individually opticallyresolvable, each polymerase enzyme complex comprising a polymeraseenzyme, a template nucleic acid, and optionally a primer hybridized tothe template nucleic acid;

sequencing reagents in contact with the surface comprising reagents forcarrying out nucleic acid synthesis including two or more of any of thelabeled nucleotide analogs described above; and

an illumination system for illuminating the polymerase enzyme complexes;

an optical detection system for detecting fluorescence from the labelednucleotide analogs while they are interacting with the polymerase enzymecomplexes; and

a computer for analyzing the signals detected by the detection system todetermine the sequential addition of nucleotides to a nucleic acidstrand complementary to a strand of the template nucleic acid.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein can be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following Examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1. Synthesis of Bis-Biotin Nucleotide Compounds

A variety of nucleotide compounds containing bis-biotin linkers havebeen synthesized for use in single-molecule real-time sequencingreactions. These compounds have been assembled with dye-labeledcompounds, or their intermediate forms, that also contain bis-biotinlinkers, using avidin proteins to create dye-labeled nucleotide analogcomplexes, for example as described in Example 2 and as illustrated inFIGS. 7A-7D and 7F. Additional examples of labeled nucleotide analogsthat have been prepared according to these methods are graphicallyillustrated in FIGS. 3E-3Z and 3A′-3O′. Many of the analogs demonstrateimproved photostability, brightness, and reaction kinetics in automatedDNA sequencing reactions involving DNA polymerase. See also U.S. PatentApplication Publication No. 2013/0316912 A1. Use of the assembledfluorescent nucleotide reagent complexes in real-time sequencingreactions is described in Example 3.

The bis-biotin-containing nucleotide reagent compounds of the instantdisclosure can include two nucleotide arms, for example as shown in FIG.26 for Control-SG1x4-dG2. As shown in this structure, each of the twonucleotide arms can contain a guanosine nucleoside, a hexaphosphatechain, a linker group, including a triazole moiety resulting from a“click” coupling reaction, and a pair of shield elements, eachcomprising two side chains (“SG1” side chains—see above reactionschemes) that each contains three anionic side chains. Such shieldelements, when incorporated into fluorescent nucleotide reagentcompounds, have been shown to prevent photodamage of the polymeraseenzyme and to provide other advantages in sequencing reactions. See,e.g., U.S. Patent Application Publication Nos. 2015/0050659 A1 and2016/0237279 A1. They have been found here to modulate the affinity ofthe nucleotide reagent for the polymerase enzyme and/or to provide otherimprovements in the kinetics of polymerase reactions using nucleotidereagents containing these groups. The exemplary Control-SG1x4-dG2compound further contains a triamino-cyclohexyl multivalent central coreelement that provides a branch point for the two nucleotide arms andthat also provides a binding site for the bis-biotin group, which itselfcomprises a triamino triazine multivalent central core element thatprovides a branch point for the bis-biotin terminal coupling element ofthe molecule.

Synthesis of the reagent compound of FIG. 26 was performed as describedgenerally in U.S. Patent Application Publication No. 2015/0050659 A1.

Alternative nucleotide reagent compounds can include just one nucleotidearm, for example as shown in FIG. 27 for Control-SG1x2-dG. In thiscompound, there is a single nucleotide arm, where the nucleoside islinked to a hexaphosphate chain, a linker group, and a pair of shieldelements, much the same as in the Control-SG1x4-dG2 dinucleotidecompound described above. Unlike the dinucleotide structure, however, inthe mononucleotide compound, the shielded nucleotide arm is coupleddirectly to the triamino triazine multivalent central core element thatcarries the bis-biotin terminal coupling element.

The variant structures illustrated in FIG. 28 also contain a singlenucleotide arm but differ from the Control-SG1x2-dG mononucleotidecompound in including an extra pair, or “layer”, of shield elements (forLayered-SG1x4-dG) or two extra pairs of shield elements (forLayered-SG1x6-dG). It should be understood that the compounds extendbeyond the terminal triazole moiety in each case to include an extrasegment of the nucleotide linker element, a linear polyphosphateelement, and a nucleoside.

Another variant mononucleotide compound contains a branch, or “split”within each of the shield elements, such that additional anionic sidechains are attached to the shield element with a branching group coupledto an aromatic group with multiple anionic side chains. The structureshown in FIG. 29, Split-SG1x4-dG, represents the complete nucleotidereagent compound, including the complete nucleotide linker, thepolyphosphate element, and the nucleoside (in this case a “dG”nucleoside).

Yet another variant structure of a mononucleotide reagent includes ananionic aromatic “spacer” group within the nucleotide arm of thereagent. An exemplary structure, DISC-SG1x2-dG, is shown in FIG. 30. Asshown, the structure includes a “dG” nucleoside attached to apolyphosphate element. It is otherwise identical to the Control-SG1x2-dGstructure shown in FIG. 27, except that it includes a1H-2,3-dihydroisoquinoline-8-sulfo-6-carboxylic acid (“DISC”) spacerelement inserted within one of the amide bonds of the nucleotide linkerof the Control-SG1x2-dG structure.

Further variant mononucleotide reagents with anionic aromatic spacergroups in their nucleotide arms include compounds comprising at leastone shield element. For example, the DISC-Split-SG1x4-dA compound shownin FIG. 3I includes the DISC group of DISC-SG1x2-dG in combination withthe split shield groups of Split-SG1x4-dG. In this particular example,the nucleoside is a deoxyadenosine (“dA”) nucleoside. The rest of themolecule, in particular the split shield element and the bis-biotingroup, is the same as Split-SG1x4-dG.

In still another variant of the shield structure in nucleotide compoundscontaining an anionic aromatic spacer group, the at least one shieldelement can include a triple-branched structure with additional anionicside chains, for example as shown in DISC-Split-SG1x6-dG of FIG. 32,thus carrying 6 of the sulfonic acid-substituted SG1 side chains.

The branching of the shield groups can be extended still further, forexample as shown in DISC-Split-SG1x12-dG of FIG. 33, where the sidechains include an additional branching element, so that they carry 12 ofthe sulfonic acid-substituted SG1 groups. All of the above-describedstructures have been assembled using known reactions, for example usingclick chemistry, copper-free click chemistry, and the like, for exampleas described in detail in U.S. Patent Application Publication No.2015/0050659 A1.

Further modifications to the instant nucleotide compounds included theincorporation of an anionic aromatic spacer group into both nucleotidelinker elements of a dinucleotide compound, for example as shown inDISC2-Split-SG1x12-dG2 of FIG. 34.

Another exemplary dinucleotide compound containing an anionic aromaticspacer group in both linker elements and 12 SG1 shield group elements isshown in FIG. 35 as DISC2-Split-SG1x12(click)-dG2. TheDISC2-Split-SG1x12(click)-dG2 and DISC2-Split-SG1x12(amide)-dG2 differin the coupling of the shield element to the nucleotide linker, and inthe orientation and linkage of the central 3,4,5-trioxybenzoyl groupwithin the linker.

The just-described alternative coupling of the shield group elements tothe nucleotide linker, and the orientation and linkage of the central3,4,5-trioxybenzoyl group within the linker, has also been compared inmononucleotide compounds, for example as shown for DISC-Split-SG1x6-dGand DISC-Split-SG1x6-dG(click) of FIGS. 36 and 37, respectively.

The nucleotide compounds described above were assembled into labelednucleotide analog complexes, for example as described below in Example2. These fluorescent nucleotide analogs were then compared in DNAsequencing reactions, for example as described below in Example 3.

Example 2. Assembly of Dye-Labeled Nucleotide Analogs

The mononucleotide and dinucleotide compounds described above have beenassembled into dye-labeled nucleotide analogs by combining thenucleotide compounds with one or more avidin proteins and one or moredye-labeled compounds or intermediates. For most of the kineticexperiments described in Example 3, the nucleotide compounds wereassembled using a single avidin protein and a simple, unshieldeddye-labeled compound such as dye-labeled compound illustratedgraphically in FIG. 4A. Such assembly can be performed as described inU.S. Patent Application Publication No. 2013/0316912 A1. More complexanalog structures have also been assembled, for example using thepathways shown in FIGS. 7A-7D and 7F. These analogs, such as the analogsdepicted in FIG. 19A, have also been assessed in kinetic sequencingassays, as described in Example 3.

Example 3. Use of the Dye-Labeled Nucleotide Analogs in Real-TimeSequencing Reactions

Single-molecule real time sequencing reactions using the fluorescentnucleotide analogs described in Example 2 were carried out in azero-mode waveguide (“ZMW”) array having 3000 discrete cores. Thereactions were observed using a highly multiplexed confocal fluorescentmicroscope providing a targeted illumination profile, e.g., a separatespot for each core. See, e.g., U.S. Pat. No. 7,714,303, which isincorporated herein by reference in its entirety for all purposes.Fluorescent signals from the various ZMWs were detected using an EMCCDcamera, and the signals were subjected to pulse recognition and basecalling processes. See, e.g., U.S. Pat. No. 8,182,993, which isincorporated herein by reference in its entirety for all purposes. Thesequencing was carried out generally as described in Eid, J. et al.(2009) Science 323:133-138, and the corresponding supplementalinformation included therewith.

For each of the sequencing reactions the laser power was 0.5 to 2.0 μW/μm² and a camera frame rate of 100 FPS. The template was a circular vD“SMRTbell” template of about 11000 kb as described in U.S. Pat. No.8,236,499, filed Mar. 27, 2009. The polymerase enzyme immobilized in thezero mode waveguide was a mutant Φ29 polymerase as described in U.S.Pat. No. 8,257,954, filed Mar. 30, 2009. The reaction mixture had aBis-Tris Propane pH 7.5 buffer, antioxidants, 40 mM DTT, 120 mM KOAc tocontrol ionic strength; 30 mM MgOAc and 4 to 8% organic solventadditive. The mixture also contained a set of nucleotide analogscorresponding to A, G, C, and T, each present at 150-400 nM, and eachhaving a unique dye-labeled compound complexed to the nucleotidecompound through an avidin protein. Ten minute to 120 minute movies ofthe sequencing reactions were obtained. Data were collected on thebrightness, kinetics (pulse width, the interpulse distance (IPD)),photophysical signal stability, sequencing error types, read length, andaccuracy.

As shown in the sequencing reactions of FIG. 12A, a simplemononucleotide analog structure results in a roughly 1% improvement inthe accuracy of the sequencing reaction (condition 1) compared to acomparable dinucleotide structure (condition 2). The data are compareddirectly in FIG. 12B, where the normalized accuracy is increased from0.893 (left plot) for the dinucleotide to 0.904 (right plot) for themononucleotide.

At the same time, as shown in FIGS. 13A and 13B, the kinetics ofincorporation are not significantly different for the mononucleotide anddinucleotide reagents for each of the four bases. By way of background,the kinetics of a single-molecule real-time sequencing reaction aregenerally described as including an observable phase, which generallycorresponds to the time period during which a particular phase isobservable. The time period for a bright phase, for example, can berepresented by the pulse width (PW) of a signal. The time period for adark phase can be represented, for example, by the interpulse distance(IPD) of a signal. The length of each time period will not be the samefor each nucleotide addition, resulting in a distribution of the lengthof these time periods. In some cases, the time periods with the shortestlength will not be detected, thus leading to errors, for example insingle-molecule sequencing. FIG. 13A shows IPD distribution curvescomparing mononucleotide analogs and dinucleotide analogs for each ofthe four bases (A, C, G, and T), where the base is indicated at the topof each panel. In these plots, the x-axis relates to detector frames,with 1 frame equal to 10 milliseconds. The y-axis represents theempirical cumulative distribution functions (ecdf), a unitless value,ranging from 0 to 1, that describes the probability of seeing the IPD ofa certain duration in frames.

Normalized IPD values for each of the conditions are provided in FIG.13B, with the dinucleotide analog on the left and the mononucleotideanalog on the right. The left-most pair reflects the cumulativenormalized IPD values for all four bases, while the following four pairsreflect the separate normalized IPD values for each indicateddeoxyribonucleotide. The dinucleotides were present at 200 nM for eachbase, and the mononucleotides were present at 250 nM for dC and at 200nM for dG, dT, and dA. As indicated by the large arrow in the comparisonof IPD distributions for dG, the mononucleotide is slightly slower thanthe dinucleotide reagent.

Variants of the mononucleotide and dinucleotide structures described inExample 1 have been tested in single-molecule real-time sequencingreactions to compare the effects of various other structural features onthe behavior of the dye-labeled nucleotide analogs in sequencingreactions. For example, FIGS. 14A and 14B illustrate the incorporationkinetics of the control analog (Control-SG1x4-dG2) (condition 1); adouble-layered analog (Layered-SG1x4-dG) (condition 2); a split sidechain analog (Split-SG1x4-dG) (condition 3); and an analog comprisingthe DISC anionic aromatic spacer (DISC-SG1x2-dG) (condition 4).

As is apparent in FIG. 14A, the kinetics of incorporation of theabove-described nucleotide reagents increase in the orderControl-SG1x4-dG2<DISC-SG1x2-dG<Layered-SG1x4-dG<Split-SG1x4-dG formononucleotides containing G. FIG. 14B provides a comparison of thenormalized IPD values for each of these reagents. As can be calculatedfrom these data, the acceleration factor relative to control are:Split-SG1x4-dG: 1.82x; DISC-SG1x2-dG: 1.42x; and Layered-SG1x4-dG:1.53x.

FIGS. 15A-15C illustrate the incorporation kinetics (normalized IPDs)(FIG. 15A), global rates (FIG. 15B), and merging errors (FIG. 15C) forthe dinucleotide control analog (Control-SG1x4-dG2) (condition 1), for amononucleotide analog with six shield groups but no anionic aromaticspacer (condition 2), for a mononucleotide analog with four shieldgroups and an anionic aromatic spacer (condition 3), and for amononucleotide analog with six shield groups and an anionic aromaticspacer (DISC-Split-SG1x6-dG) (condition 4). In each case, the reagentsare dG-nucleotide analogs.

As is apparent from the results, either including an anionic aromaticspacer group in the analogs (condition 4 vs. condition 2) or increasingthe number of shield groups in the analogs from four to six (condition 4vs. condition 3) results in improved kinetics, with the IPD valuedecreasing by approximately 20% in the analogs containing thesemodifications. Inclusion of the anionic aromatic spacer group in theanalogs also improves the global rate and accuracy of sequencing.

The nature of the anionic aromatic spacer group can also impact thebehavior of the modified nucleotide analogs in sequencing reactions.Specifically, as shown in FIGS. 16A-16C, substituting the DISC spacer ofthe DISC-Split-SG1x6-dG analog with a4,8-disulfonaphthalene-2,6-dicarboxylic acid spacer (see below) resultsin approximately 10% slower kinetics (based on IPD values) but slightlywider pulse widths.

In FIG. 16A, the normalized IPD values for analogs containing each ofthe four bases are shown for the dinucleotide control analog(Control-SG1x4-dG2) (condition 1), for the mononucleotide analog withfour shield groups and the DISC spacer group (DISC-Split-SG1x4-dG)(condition 2), for the mononucleotide analog with six shield groups andthe DISC spacer group (DISC-Split-SG1x6-dG) (condition 3), and for themononucleotide analog with six shield groups and the DSDC spacer group(condition 4). The IPD distribution curves for the G-nucleotide analogsare compared in FIG. 16B, and the normalized pulse-widths for theG-nucleotide analogs are compared in FIG. 16C.

The number of side chains in the shield elements, and thus the chargeadjacent to the nucleotide, can be further increased, for example asshown above in structure, DISC-Split-SG1x12-dG. The kinetics of ananalog containing this structure in single-molecule real-time sequencingreactions were analyzed at various concentrations, as illustrated inFIGS. 17A and 17B. In these assays, the DISC-Split-SG1x12-dG analog wasmeasured at 100 nM (condition 1), 150 nM (condition 2), or 200 nM(condition 3), and compared to DISC-Split-SG1x6-dG at 200 nM (condition4) and to Control-SG1x4-dG2 at 200 nM (condition 5). The IPDdistribution curves for these analogs and conditions are compared inFIG. 17A, and the normalized IPD values for the G-nucleotide analogs arecompared in FIG. 17B. These data indicate that doubling the charge ofthe side chains does not lead to a significant acceleration in IPD.

The anionic aromatic spacer group has additionally been incorporatedinto both linker groups of two dinucleotide analogs. Specifically,DISC2-Split-SG1x12(amide)-dG2 and DISC2-Split-SG1x12(click)-dG2, both ofwhich are shown above, contain a DISC anionic aromatic spacer group ineach of the two linker arms. Analogs containing these structures havebeen compared to the comparable triple-SG mononucleotide analog,DISC-Split-SG1x6-dG, that also contains the DISC anionic aromatic spacergroup in the nucleotide linker. Analogs containing these structures havealso been compared to the dinucleotide analog, Control-SG1x4-dG2, thatlacks the anionic aromatic spacer group in the nucleotide linkers. Asillustrated in FIG. 18, the two DISC-containing dinucleotide analogs,DISC2-Split-SG1x12(amide)-dG2 (condition 1) andDISC2-Split-SG1x12(click)-dG2 (condition 2) do not display usefullydifferent kinetics compared to the non-DISC dinucleotide analog,Control-SG1x4-dG2 (condition 4), with one showing somewhat shorter IPDvalues and the other showing somewhat longer IPD values. As seenpreviously, the DISC-containing mononucleotide analog,DISC-Split-SG1x6-dG (condition 3), displays somewhat slower kineticsthan any of the dinucleotide analogs.

FIG. 19A illustrates some additional labeled nucleotide analogstructures comprising two avidin proteins that have been assembled usingthe above-described nucleotide and dye-labeled compounds. Specifically,the SG1x2-dT_4 analog comprises a dinucleotide structure with only twoside chains per shield element and no anionic aromatic spacer element.The DISC-Split-SG1x6-dT_2 analog comprises a mononucleotide structurewith six side chains per shield element and a DISC anionic aromaticspacer element in the nucleotide linker. The DISC-Split-SG1x6-dT_4 isthe dinucleotide variant of this structure, with six side chains pershield element and the DISC anionic aromatic spacer element. FIGS. 19Band 19C show normalized IPD values and polymerization rates for ananalog comprising the DISC-Split-SG1x6-dT_4 dinucleotide variant at 100nM (condition 3), 150 nM (condition 4), and 250 nM (condition 5)concentrations compared to an analog comprising the dinucleotidestructure with two side chains and lacking an anionic aromatic spacerelement, SG1x2-dT_4, at 250 nM (condition 1), and an analog comprisingthe mononucleotide structure with six side chains and a DISC anionicaromatic spacer element, DISC-Split-SG1x6-dT_2, at 250 nM (condition 2).It is apparent from these data that, in addition to improved accuracy, amononucleotide compound with both a shield element and an anionicaromatic spacer element as affinity modulating elements has comparablekinetics to a dinucleotide compound that comprises these elements.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

What is claimed is:
 1. A nucleotide compound of structural formula (I):

wherein L is a nucleotide linker element comprising at least oneaffinity modulating element; wherein the at least one affinitymodulating element is a shield element; P is a polyphosphate element; Nuis a nucleoside element; X is a multivalent central core element; B″ isa terminal coupling element, wherein the terminal coupling elementcomprises a biotin moiety; n is an integer from 1 to 4; and o is 0 or 1.2. The nucleotide compound of claim 1, wherein the at least one affinitymodulating element decreases the K_(m) of a nucleotide-dependent enzymefor the nucleotide compound or increases the enzymatic rate of thenucleotide-dependent enzyme.
 3. The nucleotide compound of claim 1,wherein the shield element comprises a plurality of side chains.
 4. Thenucleotide compound of claim 3, wherein at least one side chain has amolecular weight of at least
 300. 5. The nucleotide compound of claim 3,wherein all of the side chains have a molecular weight of at least 300.6. The nucleotide compound of claim 3, wherein at least one side chaincomprises a negatively-charged component.
 7. The nucleotide compound ofclaim 6, wherein the negatively-charged component comprises a sulfonicacid.
 8. The nucleotide compound of claim 3, wherein at least one sidechain comprises a substituted phenyl group.
 9. The nucleotide compoundof claim 8, wherein the at least one side chain comprises the structure:

wherein each x is independently an integer from 1 to
 6. 10. Thenucleotide compound of claim 9, wherein each x is independently aninteger from 1 to
 4. 11. The nucleotide compound of claim 3, wherein atleast one side chain comprises a triazole.
 12. The nucleotide compoundof claim 3, wherein at least one side chain comprises the structure:


13. The nucleotide compound of claim 1, wherein the shield elementcomprises the structure:

wherein each y is independently an integer from 1 to
 6. 14. Thenucleotide compound of claim 1, wherein L comprises the structure:


15. The nucleotide compound of claim 1, wherein L further comprises analkyl linker group, optionally comprising an amide bond.
 16. Thenucleotide compound of claim 1, wherein L further comprises a triazole.17. The nucleotide compound of claim 1, wherein B″ comprises abis-biotin moiety.
 18. The nucleotide compound of claim 1, wherein ois
 1. 19. The nucleotide compound of claim 18, wherein n is
 2. 20. Thenucleotide compound of claim 18, wherein X comprises a polyamine moiety.21. The nucleotide compound of claim 1, wherein o is 0 and n is
 1. 22.The nucleotide compound of claim 1, wherein the compound does notcontain a dye.
 23. A nucleotide reagent composition comprising an avidinprotein and the nucleotide compound of claim
 1. 24. The nucleotidecompound of claim 1, wherein the nucleotide linker element comprisesboth an aromatic spacer element and a shield element.